Quinolines and related analogs as sirtuin modulators

ABSTRACT

Provided herein are novel sirtuin-modulating compounds and methods of use thereof. The sirtuin-modulating compounds may be used for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders that would benefit from increased mitochondrial activity. Also provided are compositions comprising a sirtuin-modulating compound in combination with another therapeutic agent.

BACKGROUND

The Silent Information Regulator (SIR) family of genes represents ahighly conserved group of genes present in the genomes of organismsranging from archaebacteria to higher eukaryotes. The encoded SIRproteins are involved in diverse processes from regulation of genesilencing to DNA repair. The proteins encoded by members of the SIR genefamily show high sequence conservation in a 250 amino acid core domain.A well-characterized gene in this family is S. cerevisiae SIR2, which isinvolved in silencing HM loci that contain information specifying yeastmating type, telomere position effects and cell aging. The yeast Sir2protein belongs to a family of histone deacetylases. The Sir2 homolog,CobB, in Salmonella typhimurium, functions as an NAD (nicotinamideadenine dinucleotide)-dependent ADP-ribosyl transferase.

The Sir2 protein is a class III deacetylase which uses NAD as acosubstrate. Unlike other deacetylases, many of which are involved ingene silencing, Sir2 is insensitive to class I and II histonedeacetylase inhibitors like trichostatin A (TSA).

Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NADhydrolysis, producing nicotinamide and a novel acetyl-ADP ribosecompound. The NAD-dependent deacetylase activity of Sir2 is essentialfor its functions which can connect its biological role with cellularmetabolism in yeast. Mammalian Sir2 homologs have NAD-dependent histonedeacetylase activity.

Biochemical studies have shown that Sir2 can readily deacetylate theamino-terminal tails of histones H3 and H4, resulting in the formationof 1-O-acetyl-ADP-ribose and nicotinamide. Strains with additionalcopies of SIR2 display increased rDNA silencing and a 30% longer lifespan. It has recently been shown that additional copies of the C.elegans SIR2 homolog, sir-2.1, and the D. melanogaster dSir2 genegreatly extend life span in those organisms. This implies that theSIR2-dependent regulatory pathway for aging arose early in evolution andhas been well conserved. Today, Sir2 genes are believed to have evolvedto enhance an organism's health and stress resistance to increase itschance of surviving adversity.

In humans, there are seven Sir2-like genes (SIRT1-SRT7) that share theconserved catalytic domain of Sir2. SIRT1 is a nuclear protein with thehighest degree of sequence similarity to Sir2. SIRT1 regulates multiplecellular targets by deacetylation including the tumor suppressor p53,the cellular signaling factor NF-KB, and the FOXO transcription factor.

SIRT3 is a homolog of SIRT1 that is conserved in prokaryotes andeukaryotes. The SIRT3 protein is targeted to the mitochondrial cristaeby a unique domain located at the N-terminus. SIRT3 has NAD+-dependentprotein deacetylase activity and is upbiquitously expressed,particularly in metabolically active tissues. Upon transfer to themitochondria, SIRT3 is believed to be cleaved into a smaller, activeform by a mitochondrial matrix processing peptidase (MPP).

Caloric restriction has been known for over 70 years to improve thehealth and extend the lifespan of mammals. Yeast life span, like that ofmetazoans, is also extended by interventions that resemble caloricrestriction, such as low glucose. The discovery that both yeast andflies lacking the SIR2 gene do not live longer when caloricallyrestricted, provides evidence that SIR2 genes mediate the beneficialhealth effects of a restricted calorie diet. Moreover, mutations thatreduce the activity of the yeast glucose-responsive cAMP (adenosine3′,5′-monophosphate)-dependent (PKA) pathway extend life span in wildtype cells but not in mutant sir2 strains, demonstrating that SIR2 islikely to be a key downstream component of the caloric restrictionpathway.

SUMMARY

Provided herein are novel sirtuin-modulating compounds and methods ofuse thereof.

In one aspect, the invention provides sirtuin-modulating compounds ofStructural Formulas (I), (II), and (III) as are described in detailbelow.

In another aspect, the invention provides methods for usingsirtuin-modulating compounds, or compostions comprisingsirtuin-modulating compounds. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be used for a variety of therapeutic applications including, forexample, increasing the lifespan of a cell, and treating and/orpreventing a wide variety of diseases and disorders including, forexample, diseases or disorders related to aging or stress, diabetes,obesity, neurodegenerative diseases, chemotherapeutic inducedneuropathy, neuropathy associated with an ischemic event, oculardiseases and/or disorders, cardiovascular disease, blood clottingdisorders, inflammation, and/or flushing, etc. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay also be used for treating a disease or disorder in a subject thatwould benefit from increased mitochondrial activity, for enhancingmuscle performance, for increasing muscle ATP levels, or for treating orpreventing muscle tissue damage associated with hypoxia or ischemia.

In other embodiments, sirtuin-modulating compounds that decrease thelevel and/or activity of a sirtuin protein may be used for a variety oftherapeutic applications including, for example, increasing cellularsensitivity to stress, increasing apoptosis, treatment of cancer,stimulation of appetite, and/or stimulation of weight gain, etc. Asdescribed further below, the methods comprise administering to a subjectin need thereof a pharmaceutically effective amount of asirtuin-modulating compound.

In certain aspects, the sirtuin-modulating compounds may be administeredalone or in combination with other compounds, including othersirtuin-modulating compounds, or other therapeutic agents.

DETAILED DESCRIPTION 1. Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule (such as a nucleicacid, an antibody, a protein or portion thereof, e.g., a peptide), or anextract made from biological materials such as bacteria, plants, fungi,or animal (particularly mammalian) cells or tissues. The activity ofsuch agents may render it suitable as a “therapeutic agent” which is abiologically, physiologically, or pharmacologically active substance (orsubstances) that acts locally or systemically in a subject.

The term “bioavailable” when referring to a compound is art-recognizedand refers to a form of a compound that allows for it, or a portion ofthe amount of compound administered, to be absorbed by, incorporated to,or otherwise physiologically available to a subject or patient to whomit is administered.

“Biologically active portion of a sirtuin” refers to a portion of asirtuin protein having a biological activity, such as the ability todeacetylate. Biologically active portions of a sirtuin may comprise thecore domain of sirtuins. Biologically active portions of SIRT1 havingGenBank Accession No. NP_(—)036370 that encompass the NAD+ bindingdomain and the substrate binding domain, for example, may includewithout limitation, amino acids 62-293 of GenBank Accession No.NP_(—)036370, which are encoded by nucleotides 237 to 932 of GenBankAccession No. NM_(—)012238. Therefore, this region is sometimes referredto as the core domain. Other biologically active portions of SIRT1, alsosometimes referred to as core domains, include about amino acids 261 to447 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 834 to 1394 of GenBank Accession No. NM_(—)012238; aboutamino acids 242 to 493 of GenBank Accession No. NP_(—)036370, which areencoded by nucleotides 777 to 1532 of GenBank Accession No.NM_(—)012238; or about amino acids 254 to 495 of GenBank Accession No.NP_(—)036370, which are encoded by nucleotides 813 to 1538 of GenBankAccession No. NM_(—)012238.

The term “companion animals” refers to cats and dogs. As used herein,the term “dog(s)” denotes any member of the species Canis familiaris, ofwhich there are a large number of different breeds. The term “cat(s)”refers to a feline animal including domestic cats and other members ofthe family Felidae, genus Felis.

“Diabetes” refers to high blood sugar or ketoacidosis, as well aschronic, general metabolic abnormalities arising from a prolonged highblood sugar status or a decrease in glucose tolerance. “Diabetes”encompasses both the type I and type II (Non Insulin Dependent DiabetesMellitus or NIDDM) forms of the disease. The risk factors for diabetesinclude the following factors: waistline of more than 40 inches for menor 35 inches for women, blood pressure of 130/85 mmHg or higher,triglycerides above 150 mg/dl, fasting blood glucose greater than 100mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50mg/dl in women.

The term “ED₅₀” is art-recognized. In certain embodiments, ED₅₀ meansthe dose of a drug which produces 50% of its maximum response or effect,or alternatively, the dose which produces a pre-determined response in50% of test subjects or preparations. The term “LD₅₀” is art-recognized.In certain embodiments, LD₅₀ means the dose of a drug which is lethal in50% of test subjects. The term “therapeutic index” is an art-recognizedterm which refers to the therapeutic index of a drug, defined asLD₅₀/ED₅₀.

The term “hyperinsulinemia” refers to a state in an individual in whichthe level of insulin in the blood is higher than normal.

The term “insulin resistance” refers to a state in which a normal amountof insulin produces a subnormal biologic response relative to thebiological response in a subject that does not have insulin resistance.

An “insulin resistance disorder,” as discussed herein, refers to anydisease or condition that is caused by or contributed to by insulinresistance. Examples include: diabetes, obesity, metabolic syndrome,insulin-resistance syndromes, syndrome X, insulin resistance, high bloodpressure, hypertension, high blood cholesterol, dyslipidemia,hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke,coronary artery disease or myocardial infarction, hyperglycemia,hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance,delayed insulin release, diabetic complications, including coronaryheart disease, angina pectoris, congestive heart failure, stroke,cognitive functions in dementia, retinopathy, peripheral neuropathy,nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome,hypertensive nephrosclerosis some types of cancer (such as endometrial,breast, prostate, and colon), complications of pregnancy, poor femalereproductive health (such as menstrual irregularities, infertility,irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy,cholesterol related disorders, such as gallstones, cholescystitis andcholelithiasis, gout, obstructive sleep apnea and respiratory problems,osteoarthritis, and prevention and treatment of bone loss, e.g.osteoporosis.

The term “livestock animals” refers to domesticated quadrupeds, whichincludes those being raised for meat and various byproducts, e.g., abovine animal including cattle and other members of the genus Bos, aporcine animal including domestic swine and other members of the genusSus, an ovine animal including sheep and other members of the genusOvis, domestic goats and other members of the genus Capra; domesticatedquadrupeds being raised for specialized tasks such as use as a beast ofburden, e.g., an equine animal including domestic horses and othermembers of the family Equidae, genus Equus.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, livestock animals (including bovines, porcines, etc.),companion animals (e.g., canines, felines, etc.) and rodents (e.g., miceand rats).

“Obese” individuals or individuals suffering from obesity are generallyindividuals having a body mass index (BMI) of at least 25 or greater.Obesity may or may not be associated with insulin resistance.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

A “patient”, “subject”, “individual” or “host” refers to either a humanor a non-human animal.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug to a host. If it is administeredprior to clinical manifestation of the unwanted condition (e.g., diseaseor other unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if administered after manifestation of the unwantedcondition, the treatment is therapeutic (i.e., it is intended todiminish, ameliorate or maintain the existing unwanted condition or sideeffects therefrom).

The term “pyrogen-free”, with reference to a composition, refers to acomposition that does not contain a pyrogen in an amount that would leadto an adverse effect (e.g., irritation, fever, inflammation, diarrhea,respiratory distress, endotoxic shock, etc.) in a subject to which thecomposition has been administered. For example, the term is meant toencompass compositions that are free of, or substantially free of, anendotoxin such as, for example, a lipopolysaccharide (LPS).

“Replicative lifespan” of a cell refers to the number of daughter cellsproduced by an individual “mother cell.” “Chronological aging” or“chronological lifespan,” on the other hand, refers to the length oftime a population of non-dividing cells remains viable when deprived ofnutrients. “Increasing the lifespan of a cell” or “extending thelifespan of a cell,” as applied to cells or organisms, refers toincreasing the number of daughter cells produced by one cell; increasingthe ability of cells or organisms to cope with stresses and combatdamage, e.g., to DNA, proteins; and/or increasing the ability of cellsor organisms to survive and exist in a living state for longer under aparticular condition, e.g., stress (for example, heatshock, osmoticstress, high energy radiation, chemically-induced stress, DNA damage,inadequate salt level, inadequate nitrogen level, or inadequate nutrientlevel). Lifespan can be increased by at least about 20%, 30%, 40%, 50%,60% or between 20% and 70%, 30% and 60%, 40% and 60% or more usingmethods described herein.

“Sirtuin-activating compound” refers to a compound that increases thelevel of a sirtuin protein and/or increases at least one activity of asirtuin protein. In an exemplary embodiment, a sirtuin-activatingcompound may increase at least one biological activity of a sirtuinprotein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplarybiological activities of sirtuin proteins include deacetylation, e.g.,of histones and p53; extending lifespan; increasing genomic stability;silencing transcription; and controlling the segregation of oxidizedproteins between mother and daughter cells.

“Sirtuin protein” refers to a member of the sirtuin deacetylase proteinfamily, or

preferably to the sir2 family, which include yeast Sir2 (GenBankAccession No. P53685), C. elegans Sir-2.1 (GenBank Accession No.NP_(—)501912), and human SIRT1 (GenBank Accession No. NM_(—)012238 andNP_(—)036370 (or AF083106)) and SIRT2 (GenBank Accession No.NM_(—)012237, NM_(—)030593, NP_(—)036369, NP_(—)085096, and AF083107)proteins. Other family members include the four additional yeastSir2-like genes termed “HST genes” (homologues of Sir two) HST1, HST2,HST3 and HST4, and the five other human homologues hSIRT3, hSIRT4,hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 andFrye et al. (1999) BBRC 260:273). Preferred sirtuins are those thatshare more similarities with SIRT1, i.e., hSIRT1, and/or Sir2 than withSIRT2, such as those members having at least part of the N-terminalsequence present in SIRT1 and absent in SIRT2 such as SIRT3 has.

“SIRT1 protein” refers to a member of the sir2 family of sirtuindeacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP_(—)501912), human SIRT1 (GenBank Accession No. NM_(—)012238 orNP_(—)036370 (or AF083106)), and human SIRT2 (GenBank Accession No.NM_(—)012237, NM_(—)030593, NP_(—)036369, NP_(—)085096, or AF083107)proteins, and equivalents and fragments thereof. In another embodiment,a SIRT1 protein includes a polypeptide comprising a sequence consistingof, or consisting essentially of, the amino acid sequence set forth inGenBank Accession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096,NP_(—)036369, or P53685. SIRT1 proteins include polypeptides comprisingall or a portion of the amino acid sequence set forth in GenBankAccession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369,or P53685; the amino acid sequence set forth in GenBank Accession Nos.NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369, or P53685 with 1to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative aminoacid substitutions; an amino acid sequence that is at least 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369, or P53685, andfunctional fragments thereof. Polypeptides of the invention also includehomologs (e.g., orthologs and paralogs), variants, or fragments, ofGenBank Accession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096,NP_(—)036369, or P53685.

“SIRT3 protein” refers to a member of the sirtuin deacetylase proteinfamily and/or to a homolog of a SIRT1 protein. In one embodiment, aSIRT3 protein includes human SIRT3 (GenBank Accession No. AAH01042,NP_(—)036371, or NP_(—)001017524) and mouse SIRT3 (GenBank Accession No.NP_(—)071878) proteins, and equivalents and fragments thereof. Inanother embodiment, a SIRT3 protein includes a polypeptide comprising asequence consisting of, or consisting essentially of, the amino acidsequence set forth in GenBank Accession Nos. AAH01042, NP_(—)036371,NP_(—)001017524, or NP_(—)071878. SIRT3 proteins include polypeptidescomprising all or a portion of the amino acid sequence set forth inGenBank Accession AAH01042, NP_(—)036371, NP_(—)001017524, orNP_(—)071878; the amino acid sequence set forth in GenBank AccessionNos. AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878 with 1 toabout 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acidsubstitutions; an amino acid sequence that is at least 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878, and functionalfragments thereof. Polypeptides of the invention also include homologs(e.g., orthologs and paralogs), variants, or fragments, of GenBankAccession Nos. AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878.In one embodiment, a SIRT3 protein includes a fragment of SIRT3 proteinthat is produced by cleavage with a mitochondrial matrix processingpeptidase (MPP) and/or a mitochondrial intermediate peptidase (MIP).

The terms “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” areart-recognized and refer to the administration of a subject composition,therapeutic or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes.

The term “therapeutic agent” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. The term also means any substance intended for use in thediagnosis, cure, mitigation, treatment or prevention of disease or inthe enhancement of desirable physical or mental development and/orconditions in an animal or human.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. The therapeuticallyeffective amount of such substance will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art. For example, certain compositions described herein may beadministered in a sufficient amount to produce a desired effect at areasonable benefit/risk ratio applicable to such treatment.

“Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease.

The term “vision impairment” refers to diminished vision, which is oftenonly partially reversible or irreversible upon treatment (e.g.,surgery). Particularly severe vision impairment is termed “blindness” or“vision loss”, which refers to a complete loss of vision, vision worsethan 20/200 that cannot be improved with corrective lenses, or a visualfield of less than 20 degrees diameter (10 degrees radius).

2. Sirtuin Modulators

In one aspect, the invention provides novel sirtuin-modulating compoundsfor treating and/or preventing a wide variety of diseases and disordersincluding, for example, diseases or disorders related to aging orstress, diabetes, obesity, neurodegenerative diseases, ocular diseasesand disorders, cardiovascular disease, blood clotting disorders,inflammation, cancer, and/or flushing, etc. Sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused for treating a disease or disorder in a subject that would benefitfrom increased mitochondrial activity, for enhancing muscle performance,for increasing muscle ATP levels, or for treating or preventing muscletissue damage associated with hypoxia or ischemia. Other compoundsdisclosed herein may be suitable for use in a pharmaceutical compositionand/or one or more methods disclosed herein.

In one embodiment, sirtuin-modulating compounds of the invention arerepresented by Structural Formula (I):

or a salt thereof, wherein:each of Z¹, Z², Z³, and Z⁴ is independently selected from N and CR,wherein R is selected from hydrogen, halo, —OH, fluoro-substituted C₁-C₂alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —S—(C₁-C₂)fluoro-substituted alkyl, C₁-C₄ alkyl, —O—(C₁-C₄) alkyl, —S—(C₁-C₄)alkyl and C₃-C₇ cycloalkyl;

Y is selected from N and CR³, wherein R³ is selected from hydrogen,halo, —(C₁-C₄) alkyl, —O—(C₁-C₄) alkyl, and —O—(C₁-C₂)fluoro-substituted alkyl;

no more than two of Z¹, Z², Z³, Z⁴, and Y are N;

X is selected from —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†,—NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—C(═O)O-†,t-NH—C(═O)O—NH—C(═O)NH-†, —NH—NR⁵-†, —NR⁵—NH-†, —NHO-†, —NH—CR⁵R⁶-†,—CR⁵R⁶—NH-†, —NH—C(═NR⁵)-t and —C(═NR⁵)—NH-†, wherein

-   -   † represents where X is bound to R¹, and    -   R⁵ and R⁶ are independently selected from hydrogen, C₁-C₃ alkyl,        CF₃, and (C₁-C₂ alkyl)-CF₃;

R¹ is selected from a carbocycle and a heterocycle, wherein R¹ isoptionally substituted with one to two substitutents independentlyselected from halo, C₁-C₃ alkyl, C₃-C₇ cycloalkyl, —O—R⁴, —S—R⁴,—(C₁-C₂) fluoro-substituted alkyl, —NH—CH₂—CH(OH)—CH₂OH,—O—CH₂—CH(OH)—CH₂OH, —(C₁-C₂ alkyl)-N(R⁴)(R⁴), —N(R⁴)(R⁴), —O—(C₁-C₂alkyl)-N(R⁴)(R⁴), —(C₁-C₂ alkyl)-O—(C₁-C₂ alkyl)-N(R⁴)(R⁴),—C(O)—N(R⁴)(R⁴), and —(C₁-C₂ alkyl)-C(O)—N(R⁴)(R⁴), and when R¹ isphenyl, R¹ is also optionally substituted with 3,4-methylenedioxy,fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, orfluoro-substituted 3,4-ethylenedioxy, wherein

each R⁴ is independently selected from hydrogen, and —C₁-C₄ alkyl; or

-   -   two R⁴ are taken together with the nitrogen atom to which they        are bound to form a 4- to 8-membered saturated heterocycle        optionally comprising one additional heteroatom selected from N,        S, S(═O), S(═O)₂, and O, wherein the alkyl is optionally        substituted with one or more —OH, fluoro, —NH₂, —NH(C₁-C₄        alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂        and the saturated heterocycle is optionally substituted at a        single carbon atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂,        —NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or        —N(CH₂CH₂OCH₃)₂; or    -   X and R¹ are taken together to form ring A:

or ring B:

wherein each of Z⁵, Z⁶, Z⁷, Z⁸ and Z⁹ is independently selected from CR⁷and N, wherein not more than one of Z^(S), Z⁶, Z⁷, Z⁸ and Z⁹ in ring Bis N;

each R⁷ is independently selected from hydrogen, halo, C₁-C₄ alkyl,—O—(C₁-C₃)alkyl, —O—CF₃, C₃-C₇ cycloalkyl, phenyl, and heterocyclyl,wherein the phenyl or heterocyclyl is optionally substituted with onesubstituent selected from halo, C₁-C₃ alkyl, —O—(C₁-C₃) alkyl,—S—(C₁-C₃) alkyl, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂)fluoro-substituted alkyl and —S—(C₁-C₂) fluoro-substituted alkyl; and

R² is selected from a carbocycle and a heterocycle bound to the rest ofthe compound through a carbon ring atom, wherein R² is optionallysubstituted with one to two substitutents independently selected fromhalo, —C≡N, C₁-C₃ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substitutedalkyl, —O—R⁴, —S—R⁴, —NH—CH₂—CH(OH)—CH₂OH, —O—CH₂—CH(OH)—CH₂OH, —(C₁-C₂alkyl)-N(R⁴)(R⁴), —N(R⁴)(R⁴), —O—(C₁-C₂ alkyl)-N(R⁴)(R⁴), —(C₁-C₂alkyl)-O—(C₁-C₂ alkyl)-N(R⁴)(R⁴), —C(O)—N(R⁴)(R⁴), —(C₁-C₂alkyl)-C(O)—N(R⁴)(R⁴), —O-phenyl, phenyl, and a second heterocycle, andwhen R² is phenyl, R² is also optionally substituted with3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy,3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, wherein anyphenyl or second heterocycle substituent of R² is optionally substitutedwith halo; —C≡N; C₁-C₃ alkyl, C₁-C₂ fluoro-substituted alkyl, —O—(C₁-C₂)fluoro-substituted alkyl, —O—(C₁-C₃) alkyl, —S—(C₁-C₃) alkyl, —S—(C₁-C₂)fluoro-substituted alkyl, —NH—(C₁-C₃) alkyl and —N—(C₁-C₃)₂ alkyl;

wherein the compound is not:

or

In certain embodiments, X is selected from —NH—C(═O)-†, —C(═O)—NH-†,—NH—C(═S)-†, —C(═S)—NH-†, —NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†,—NH—C(═O)O-†, —NH—C(═O)NH-†, —NH—NR⁵-†, —NR⁵—NH-†, —O—NH-†, —NH—O-†,—NH—CR⁵R⁶-†, —CR⁵R⁶—NH-†, —NH—C(═NR⁵)-†, —C(═NR⁵)—NH-†, where †represents where X is bound to R¹, and R⁵ and R⁶ are independentlyselected from hydrogen, C₁-C₃ alkyl, CF₃, and (C₁-C₂ alkyl)-CF₃.

In certain embodiments, R² is selected from a carbocycle and amonocyclic heterocycle bound to the rest of the compound through acarbon ring atom, wherein R² is optionally substituted with one to twosubstitutents independently selected from halo, —C≡N, C₁-C₃ alkyl, C₃-C₇cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R⁴, —S—R⁴,—NH—CH₂—CH(OH)—CH₂OH, —O—CH₂—CH(OH)—CH₂OH, alkyl)-N(R⁴)(R⁴), —N(R⁴)(R⁴),—O—(C₁-C₂ alkyl)-N(R⁴)(R⁴), —(C₁-C₂ alkyl)-O—(C₁-C₂ alkyl)-N(R⁴)(R⁴),—C(O)—N(R⁴)(R⁴), —(C₁-C₂ alkyl)-C(O)—N(R⁴)(R⁴), —O-phenyl, phenyl, and asecond heterocycle, and when R² is phenyl, R² is also optionallysubstituted with 3,4-methylenedioxy, fluoro-substituted3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted3,4-ethylenedioxy, wherein any phenyl or second heterocycle substituentof R² is optionally substituted with halo; —C≡N; C₁-C₃ alkyl, C₁-C₂fluoro-substituted alkyl, —O—(C₁-C₂) fluoro-substituted alkyl,—O—(C₁-C₃) alkyl, —S—(C₁-C₃) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl,—NH—(C₁-C₃) alkyl and —N—(C₁-C₃)₂ alkyl. In certain embodiments, R² hasone of these values and X has one of the values described in theprevious paragraph.

In certain embodiments, the compound of Formula (I) is represented byany one of:

In certain embodiments, the compound of Formula (I) is represented by:

In certain embodiments, the compound of Formula (I) is represented by:

In certain embodiments, X is selected from —NH—C(═O)-†, —C(═O)—NH-†,—NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-† and —NH—S(═O)₂-†. In certainembodiments, X is selected from —NH—C(═O)-f, —C(═O)—NH-† In certainembodiments, X is —C(═O)—NH-†.

In certain embodiments, X and R¹ are taken together to form ring A. Inexemplary embodiments, ring A is selected from a substituted orunsubstituted ring such as pyrrole, pyrazole, triazole and tetrazole. Incertain embodiments, X and R¹ are taken together to form ring B. Inexemplary embodiments, ring B is selected from a substituted orunsubstituted ring such as indole, indazole, and azaindole.

In certain embodiments, R¹ is selected from heterocycles comprising oneor more heteroatoms selected from N, O and S. In particular embodiments,R¹ is selected from heterocycles comprising one or two nitrogens. Inparticular embodiments, R¹ is selected from heterocycles comprising upto three heteroatoms selected from S and N. In other embodiments, R¹ isselected from heterocycles comprising up to three heteroatoms selectedfrom O and N. In certain embodiments, R¹ is selected from:

In certain embodiments, R¹ is selected from:

In certain embodiments, R² is selected from aryl and heteroaryl. Incertain such embodiments, R² is selected from:

In particular embodiments, R² is meta-substituted relative to theattachment of R² to the rest of the compound, and wherein R² isoptionally further substituted as described above. In certainembodiments, R² is selected from:

In certain embodiments, the compounds of the invention are representedby Structural Formula (II):

wherein:

X is selected from —NH—C(═O)-† and —C(═O)—NH-†;

R¹ is selected from a carbocycle and a heterocycle, wherein R¹ isoptionally substituted with one to two substitutents independentlyselected from halo, —C≡N, C₁-C₃ alkyl, C₃-C₇ cycloalkyl,fluoro-substituted C₁-C₂ alkyl, —O—R⁴, —S—R⁴, —NH—CH₂—CH(OH)—CH₂OH,—O—CH₂—CH(OH)—CH₂OH, —(C₁-C₂ alkyl)-N(R⁴)(R⁴), —N(R⁴)(R⁴), —O—(C₁-C₂alkyl)-N(R⁴)(R⁴), —(C₁-C₂ alkyl)-O—(C₁-C₂ alkyl)-N(R⁴)(R⁴),—C(O)—N(R⁴)(R⁴), and —(C₁-C₂ alkyl)-C(O)—N(R⁴)(R⁴), and when R¹ isphenyl, R¹ is also optionally substituted with 3,4-methylenedioxy,fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, orfluoro-substituted 3,4-ethylenedioxy, wherein

-   -   each R⁴ is independently selected from hydrogen, and —C₁-C₄        alkyl; or    -   two R⁴ are taken together with the nitrogen atom to which they        are bound to form a 4- to 8-membered saturated heterocycle        optionally comprising one additional heteroatom selected from N,        S, S(═O), S(═O)₂, and O, wherein the alkyl is optionally        substituted with one or more —OH, fluoro, —NH₂, —NH(C₁-C₄        alkyl), N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂ and        the saturated heterocycle is optionally substituted at a single        carbon atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂, —NH(C₁-C₄        alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂;        and

R² is phenyl optionally substituted with halo, —C≡N, C₁-C₃ alkyl, C₃-C₇cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R⁴, —S—R⁴,—NH—CH₂—CH(OH)—CH₂OH, —O—CH₂—CH(OH)—CH₂OH, —(C₁-C₂ alkyl)-N(R⁴)(R⁴),—N(R⁴)(R⁴), —O—(C₁-C₂ alkyl)-N(R⁴)(R⁴), —(C₁-C₂ alkyl)-O—(C₁-C₂alkyl)-N(R⁴)(R⁴), —C(O)—N(R⁴)(R⁴), —(C₁-C₂ alkyl)-C(O)—N(R⁴)(R⁴),—O-phenyl, phenyl, and a second heterocycle, and when R² is phenyl, R²is also optionally substituted with 3,4-methylenedioxy,fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, orfluoro-substituted 3,4-ethylenedioxy, wherein any phenyl or secondheterocycle substituent of R² is optionally substituted with halo; —C≡N;C₁-C₃ alkyl, C₁-C₂ fluoro-substituted alkyl, —O—(C₁-C₂)fluoro-substituted alkyl, —O—(C₁-C₃) alkyl, —S—(C₁-C₃) alkyl, —S—(C₁-C₂)fluoro-substituted alkyl, —NH—(C₁-C₃) alkyl and —N—(C₁-C₃)₂ alkyl.

In an alternate embodiment, the invention provides a compoundrepresented by Structural Formula III:

(III), or a salt thereof, wherein:

each of Z¹¹, Z¹², Z¹³, and Z¹⁴ is independently selected from N and CR,wherein R is selected from hydrogen, halo, —OH, —C≡N, fluoro-substitutedC₁-C₂ alkyl, —O—(C₁-C₂ fluoro-substituted alkyl), —S—(C₁-C₂fluoro-substituted alkyl), C₁-C₄ alkyl, —(C₁-C₂ alkyl)-N(R¹⁴)(R¹⁴),—O—CH₂CH(OH)CH₂OH, —O—(C₁-C₄) alkyl, —O—(C₁-C₃) alkyl-N(R¹⁴)(R¹⁴),—N(R¹⁴)(R¹⁴), —S—(C₁-C₄) alkyl and C₃-C₇ cycloalkyl;

Y is selected from N and CR¹³, wherein R¹³ is selected from hydrogen,halo, —C₁-C₄ alkyl, —O—(C₁-C₄ alkyl), and —O—(C₁-C₂ fluoro-substitutedalkyl);

no more than two of Z¹¹, Z¹², Z¹³, Z¹⁴, and Y are N;

X is selected —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†,—NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—S(O)₂—NR¹⁵-†,—NR¹⁵—S(O)₂—NH-†, —NH—C(═O)O-†, O—C(═O)—NH-†, —NH—C(═O)NH-†,—NH—C(═O)NR¹⁵-†, —NR¹⁵—C(═O)NH-†, —NH—NR¹⁵-†, —NR¹⁵—NH-†, —O—NH-†,—NH—O-†, —NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH-†, —NH—C(═NR¹⁵)-†, —C(═NR¹⁵)—NH-†,—C(═O)—NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH—C(O)-†, —NH—C(═S)—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—C(═S)—NH-†, —NH—S(O)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)—NH-†,—NH—S(O)₂—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—O—C(═O)—NH-†, —NH—C(═O)—NR¹⁴—CR¹⁵R¹⁶-†, —NH—C(═O)—CR¹⁵R¹⁶-†,and —CR'⁵R¹⁶—NH—C(═O)—O-†, wherein

† represents where X is bound to R¹¹, and:

R¹⁵ and R¹⁶ are independently selected from hydrogen, C₁-C₄ alkyl, CF₃,and —(C₁-C₄ alkyl)-CF₃;

R¹¹ is selected from a carbocycle and a heterocycle, wherein R¹¹ isoptionally substituted with one to two substitutents independentlyselected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₄fluoro-substituted alkyl, ═O, —O—R¹⁴, —S—R¹⁴, —(C₁-C₄alkyl)-N(R¹⁴)(R¹⁴), —NR¹⁴)(R¹⁴), —O—(C₂-C₄ alkyl)-N(R¹⁴)(R¹⁴),—C(O)—N(R′⁴)(R¹⁴), —C(O)—O—R¹⁴, and —(C₁-C₄ alkyl)-C(O)—N(R¹⁴)(R¹⁴), andwhen R¹¹ is phenyl, R¹¹ is also optionally substituted with3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy,3,4-ethylenedioxy, fluoro-substituted 3,4-ethylenedioxy, 0-(saturatedheterocycle), fluoro-substituted —O-(saturated heterocycle), and

C₁-C₄ alkyl-substituted 0-(saturated heterocycle), wherein

each R¹⁴ is independently selected from hydrogen, and —C₁-C₄ alkyl; or

two R¹⁴ are taken together with the nitrogen atom to which they arebound to form a 4- to 8-membered saturated heterocycle optionallycomprising one additional heteroatom selected from N, S, S(═O), S(═O)₂,and O, wherein:

-   -   when R¹⁴ is alkyl, the alkyl is optionally substituted with one        or more —OH, —O—(C₁-C₄ alkyl), fluoro, —NH₂, —NH(C₁-C₄ alkyl),        —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂ and    -   when two R¹⁴ are taken together with the nitrogen atom to which        they are bound to form a 4- to 8-membered saturated heterocycle,        the saturated heterocycle is optionally substituted at a carbon        atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂, —NH(C₁-C₄ alkyl),        —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂; and        optionally substituted at any substitutable nitrogen atom with        —C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, or —(CH₂)₂—O—CH₃;        and

R¹² is selected from a carbocycle and a heterocycle bound to the rest ofthe compound through a carbon ring atom, wherein R¹² is optionallysubstituted with one to two substitutents independently selected fromhalo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substitutedalkyl, —O—R¹⁴, —S(O)—R¹⁴, —S(O)₂—R¹⁴, —(C₁-C₄ alkyl)-N(R¹⁴)(R¹⁴),—N(R¹⁴)(R¹⁴), —O—(C₂-C₄ alkyl)-N(R¹⁴)(R¹⁴), —C(O)—N(R¹⁴)(R¹⁴), —(C₁-C₄alkyl)-C(O)—N(R¹⁴)(R¹⁴), —O-phenyl, phenyl, and a second heterocycle,and when R¹² is phenyl, R¹² is also optionally substituted with3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy,3,4-ethylenedioxy, fluoro-substituted 3,4-ethylenedioxy, or—O-(saturated heterocycle) wherein any phenyl, saturated heterocycle orsecond heterocycle substituent of R¹² is optionally substituted withhalo; —C≡N; C₁-C₄ alkyl, C₁-C₂ fluoro-substituted alkyl, —O—(C₁-C₂fluoro-substituted alkyl), —O—(C₁-C₄ alkyl), —S—(C₁-C₄ alkyl), —S—(C₁-C₂fluoro-substituted alkyl), —NH—(C₁-C₄ alkyl) and —N—(C₁-C₄ alkyl)₂,wherein:

when Z¹¹ and Z¹³ are N, Z¹² is, C—N(R¹⁴)(R¹⁴), R¹¹ is phenyl, pyridyl orthienyl and R¹² is phenyl substituted with at least one halo or —OR¹⁴,then X is not —NH—CR¹⁵R¹⁶-†; and

wherein the compound is not:

In certain embodiments of Formula III:

X is selected —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†,—NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(O)₂—NR¹⁵-†,—NR¹⁵—S(O)₂—NH-†, —NH—C(═O)O-†, O—C(═O)—NH-†, —NH—C(═O)NH-†,—NH—C(═O)NR¹⁵-†, —NR¹⁵—C(═O)NH-†, —NH—NR¹⁵-†, —NR¹⁵—NH-†, —O—NH-†,—NH—O-†, —CR¹⁵R¹⁶—NH-†, —NH—C(═NR¹⁵)-†, —C(═NR¹⁵)—NH-†,—CR¹⁵R¹⁶—NH—C(O)-†, —NH—C(═S)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—C(═S)—NH-†,—NH—S(O)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)—NH-†, —NH—S(O)₂—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—O—C(═O)—NH-†,—NH—C(═O)—NR¹⁴—CR¹⁵R¹⁶-†, —NH—C(═O)—CR¹⁵R¹⁶-†, and—CR¹⁵R¹⁶—NH—C(═O)—O-†; and

R¹² is selected from a carbocycle and a monocyclic heterocycle bound tothe rest of the compound through a carbon ring atom, wherein R¹² isoptionally substituted with one to two substitutents independentlyselected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂fluoro-substituted alkyl, —O—R¹⁴, —S—R¹⁴, —S(O)—R¹⁴, —S(O)₂—R¹⁴, —(C₁-C₄alkyl)-N(R¹⁴)(R¹⁴), —N(R¹⁴)(R¹⁴), —O—(C₂-C₄ alkyl)-N(R¹⁴)(R¹⁴),—C(O)—N(R¹⁴)(R¹⁴), (C₁-C₄ alkyl)-C(O)—N(R¹⁴)(R¹⁴), —O-phenyl, phenyl,and a second heterocycle, and when R¹² is phenyl, R¹² is also optionallysubstituted with 3,4-methylenedioxy, fluoro-substituted3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted3,4-ethylenedioxy, or —O-(saturated heterocycle) wherein any phenyl,saturated heterocycle or second heterocycle substituent of R¹² isoptionally substituted with halo; —C≡N; C₁-C₄ alkyl, C₁-C₂fluoro-substituted alkyl, —O—(C₁-C₂ fluoro-substituted alkyl), —O—(C₁-C₄alkyl), —S—(C₁-C₄ alkyl), —S—(C₁-C₂ fluoro-substituted alkyl),—NH—(C₁-C₄ alkyl) and —N—(C₁-C₄ alkyl)₂, In a more specific aspect ofthis embodiment X is selected from —NH—C(═O)-† or —C(═O)—NH-†.

In an alternate embodiment of Formula III:

X is —NH—CR¹⁵R¹⁶-†; and either

(i) at least one of Z¹¹, Z¹², Z¹³, Z¹⁴ or Y is N; or

(ii) at least one of R¹¹ or R¹² is an optionally substitutedheterocyclyl or a optionally substituted saturated carbocyclyl.

In still another embodiment of Formula III, R¹² is selected from aryland heteroaryl. In one aspect of this embodiment, R¹² is selected from:

wherein R¹² is optionally further substituted.

In still another embodiment of Formula III, R¹¹ is selected from:

wherein R¹ is optionally further substituted.

In certain embodiments of Formula III, the compound is represented by aStructural Formulae selected from:

In a more specific aspect of this embodiment, the compound isrepresented by Structural Formulae selected from IIIa, IIIi, IIIj, IIIk,or IIIl. In an even more specific aspect of this embodiment, thecompound is represented by Structural Formula IIIa.

In one specific embodiment of Formula III, the compound is representedby Structural Formula IV:

or a salt thereof, wherein:

X is selected from —NH—C(═O)-† or —C(═O)—NH-†;

R¹¹ is selected from:

wherein R¹ is optionally further substituted; and

R¹² is selected from phenyl and pyridyl, wherein R¹² is optionallysubstituted with one to two substitutents independently selected fromhalo, C₁-C₄ alkyl, C₁-C₂ fluoro-substituted alkyl, —O—R¹⁴, —S(O)₂—R¹⁴,—(C₁-C₄ alkyl)-N(R¹⁴)(R¹⁴), and —N(R¹⁴)(R¹⁴), and when R¹² is phenyl,R¹² is also optionally substituted with 3,4-methylenedioxy, orO-(saturated heterocycle).

Compounds of the invention, including novel compounds of the invention,can also be used in the methods described herein.

The compounds and salts thereof described herein also include theircorresponding hydrates (e.g., hemihydrate, monohydrate, dihydrate,trihydrate, tetrahydrate) and solvates. Suitable solvents forpreparation of solvates and hydrates can generally be selected by askilled artisan.

The compounds and salts thereof can be present in amorphous orcrystalline (including co-crystalline and polymorph) forms.

Sirtuin-modulating compounds of the invention advantageously modulatethe level and/or activity of a sirtuin protein, particularly thedeacetylase activity of the sirtuin protein.

Separately or in addition to the above properties, certainsirtuin-modulating compounds of the invention do not substantially haveone or more of the following activities: inhibition of PI3-kinase,inhibition of aldoreductase, inhibition of tyrosine kinase,transactivation of EGFR tyrosine kinase, coronary dilation, orspasmolytic activity, at concentrations of the compound that areeffective for modulating the deacetylation activity of a sirtuin protein(e.g., such as a SIRT1 and/or a SIRT3 protein).

Carbocyclic includes 5-7 membered monocyclic and 8-12 membered bicyclicrings wherein the monocyclic or bicyclic rings are selected fromsaturated, unsaturated and aromatic. Exemplary carbocycles includecyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl and naphthyl.

Heterocyclic includes 4-7 membered monocyclic and 8-12 membered bicyclicrings comprising one or more heteroatoms selected from, for example, N,O, and S atoms. In certain embodiments, the heterocyclic group isselected from saturated, unsaturated or aromatic.

Monocyclic rings include 5-7 membered aryl or heteroaryl, 3-7 memberedcycloalkyl, and 5-7 membered non-aromatic heterocyclyl. Exemplarymonocyclic groups include substituted or unsubstituted heterocycles suchas thiazolyl, oxazolyl, oxazinyl, thiazinyl, dithianyl, dioxanyl,isoxazolyl, isothiozolyl, triazolyl, furanyl, tetrahydrofuranyl,dihydrofuranyl, pyranyl, tetrazolyl, pyrazolyl, pyrazinyl, pyridazinyl,imidazolyl, pyridinyl, pyrrolyl, dihydropyrrolyl, pyrrolidinyl,thiazinyl, oxazinyl, piperidinyl, piperazinyl, pyrimidinyl, morpholinyl,tetrahydrothiophenyl, thiophenyl, cyclohexyl, cyclopentyl, cyclopropyl,cyclobutyl, cycloheptanyl, azetidinyl, oxetanyl, thiiranyl, oxiranyl,aziridinyl, and thiomorpholinyl.

Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrroyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include benzothienyl, benzofuryl,indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole,quinolinyl, isoquinolinyl and isoindolyl.

Fluoro-substituted includes from one fluoro substituent up toper-fluoro-substitution. Exemplary fluoro-substituted C₁-C₂ alkylincludes —CFH₂, CF₂H, —CF₃, —CH₂CH₂F, —CH₂CHF₂, —CHFCH₃, —CF₂CHF₂.Per-fluoro-substituted C₁-C₂ alkyl, for example, includes —CF₃, and—CF₂CF₃.

Suitable substituents on moieties indicated as being substituted orunsubstituted are those which do not substantially interfere with theability of the disclosed compounds to have one or more of the propertiesdisclosed herein. A substituent substantially interferes with theproperties of a compound when the magnitude of the property is reducedby more than about 50% in a compound with the substituent compared witha compound without the substituent.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. As usedherein, the term “stable” refers to compounds that possess stabilitysufficient to allow manufacture and that maintain the integrity of thecompound for a sufficient period of time to be useful for the purposesdetailed herein.

The compounds disclosed herein also include partially and fullydeuterated variants. In certain embodiments, one or more deuterium atomsare present for kinetic studies. One of ordinary skill in the art canselect the sites at which such deuterium atoms are present.

Also included in the present invention are salts, particularlypharmaceutically acceptable salts, of the sirtuin-modulating compoundsdescribed herein. The compounds of the present invention that possess asufficiently acidic, a sufficiently basic, or both functional groups,can react with any of a number of inorganic bases, and inorganic andorganic acids, to form a salt. Alternatively, compounds that areinherently charged, such as those with a quaternary nitrogen, can form asalt with an appropriate counterion (e.g., a halide such as bromide,chloride, or fluoride, particularly bromide).

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

According to another embodiment, the present invention provides methodsof producing the above-defined sirtuin-modulating compounds. Thecompounds may be synthesized using conventional techniques.Advantageously, these compounds are conveniently synthesized fromreadily available starting materials.

Synthetic chemistry transformations and methodologies useful insynthesizing the sirtuin-modulating compounds described herein are knownin the art and include, for example, those described in R. Larock,Comprehensive Organic Transformations (1989); T. W. Greene and p. G. M.Wuts, Protective Groups in Organic Synthesis, 2d. Ed. (1991); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis (1995).

In an exemplary embodiment, a sirtuin-modulating compound may traversethe cytoplasmic membrane of a cell. For example, a compound may have acell-permeability of at least about 20%, 50%, 75%, 80%, 90% or 95%.

Sirtuin-modulating compounds described herein may also have one or moreof the following characteristics: the compound may be essentiallynon-toxic to a cell or subject; the sirtuin-modulating compound may bean organic molecule or a small molecule of 2000 amu or less, 1000 amu orless; a compound may have a half-life under normal atmosphericconditions of at least about 30 days, 60 days, 120 days, 6 months or 1year; the compound may have a half-life in solution of at least about 30days, 60 days, 120 days, 6 months or 1 year; a sirtuin-modulatingcompound may be more stable in solution than resveratrol by at least afactor of about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 50 fold or 100fold; a sirtuin-modulating compound may promote deacetylation of the DNArepair factor Ku70; a sirtuin-modulating compound may promotedeacetylation of RelA/p65; a compound may increase general turnoverrates and enhance the sensitivity of cells to TNF-induced apoptosis.

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to inhibit a histone deacetylase (HDACs) class I, aHDAC class II, or HDACs I and II, at concentrations (e.g., in vivo)effective for modulating the deacetylase activity of the sirtuin. Forinstance, in preferred embodiments the sirtuin-modulating compound is asirtuin-activating compound and is chosen to have an EC₅₀ for activatingsirtuin deacetylase activity that is at least 5 fold less than the EC₅₀for inhibition of an HDAC I and/or HDAC II, and even more preferably atleast 10 fold, 100 fold or even 1000 fold less. Methods for assayingHDAC I and/or HDAC II activity are well known in the art and kits toperform such assays may be purchased commercially. See e.g., BioVision,Inc. (Mountain View, Calif.; world wide web at biovision.com) and ThomasScientific (Swedesboro, N.J.; world wide web at tomassci.com).

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to modulate sirtuin homologs. In one embodiment, anactivator of a human sirtuin protein may not have any substantialability to activate a sirtuin protein from lower eukaryotes,particularly yeast or human pathogens, at concentrations (e.g., in vivo)effective for activating the deacetylase activity of human sirtuin. Forexample, a sirtuin-activating compound may be chosen to have an EC₅₀ foractivating a human sirtuin, such as SIRT1 and/or SIRT3, deacetylaseactivity that is at least 5 fold less than the EC₅₀ for activating ayeast sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), andeven more preferably at least 10 fold, 100 fold or even 1000 fold less.In another embodiment, an inhibitor of a sirtuin protein from lowereukaryotes, particularly yeast or human pathogens, does not have anysubstantial ability to inhibit a sirtuin protein from humans atconcentrations (e.g., in vivo) effective for inhibiting the deacetylaseactivity of a sirtuin protein from a lower eukaryote. For example, asirtuin-inhibiting compound may be chosen to have an IC₅₀ for inhibitinga human sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity thatis at least 5 fold less than the IC₅₀ for inhibiting a yeast sirtuin,such as Sir2 (such as Candida, S. cerevisiae, etc.), and even morepreferably at least 10 fold, 100 fold or even 1000 fold less.

In certain embodiments, a sirtuin-modulating compound may have theability to modulate one or more sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,or SIRT7. In one embodiment, a sirtuin-modulating compound has theability to modulate both a SIRT1 and a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT1. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT1deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In one embodiment, a SIRT1 modulator does not have anysubstantial ability to modulate a SIRT3 protein.

In other embodiments, a SIRT3 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT3. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT3deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In one embodiment, a SIRT3 modulator does not have anysubstantial ability to modulate a SIRT1 protein.

In certain embodiments, a sirtuin-modulating compound may have a bindingaffinity for a sirtuin protein of about 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, 10⁻¹²M orless. A sirtuin-modulating compound may reduce (activator) or increase(inhibitor) the apparent Km of a sirtuin protein for its substrate orNAD+ (or other cofactor) by a factor of at least about 2, 3, 4, 5, 10,20, 30, 50 or 100. In certain embodiments, Km values are determinedusing the mass spectrometry assay described herein. Preferred activatingcompounds reduce the Km of a sirtuin for its substrate or cofactor to agreater extent than caused by resveratrol at a similar concentration orreduce the Km of a sirtuin for its substrate or cofactor similar to thatcaused by resveratrol at a lower concentration. A sirtuin-modulatingcompound may increase the Vmax of a sirtuin protein by a factor of atleast about 2, 3, 4, 5, 10, 20, 30, 50 or 100. A sirtuin-modulatingcompound may have an ED50 for modulating the deacetylase activity of aSIRT1 and/or SIRT3 protein of less than about 1 nM, less than about 10nM, less than about 100 nM, less than about 1 μM, less than about 10 μM,less than about 100 μM, or from about 1-10 nM, from about 10-100 nM,from about 0.1-1 μM, from about 1-10 μM or from about 10-100 Asirtuin-modulating compound may modulate the deacetylase activity of aSIRT1 and/or SIRT3 protein by a factor of at least about 5, 10, 20, 30,50, or 100, as measured in a cellular assay or in a cell based assay. Asirtuin-activating compound may cause at least about 10%, 30%, 50%, 80%,2 fold, 5 fold, 10 fold, 50 fold or 100 fold greater induction of thedeacetylase activity of a sirtuin protein relative to the sameconcentration of resveratrol. A sirtuin-modulating compound may have anED50 for modulating SIRT5 that is at least about 10 fold, 20 fold, 30fold, 50 fold greater than that for modulating SIRT1 and/or SIRT3.

3. Exemplary Uses

In certain aspects, the invention provides methods for modulating thelevel and/or activity of a sirtuin protein and methods of use thereof.

In certain embodiments, the invention provides methods for usingsirtuin-modulating compounds wherein the sirtuin-modulating compoundsactivate a sirtuin protein, e.g., increase the level and/or activity ofa sirtuin protein. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for a variety oftherapeutic applications including, for example, increasing the lifespanof a cell, and treating and/or preventing a wide variety of diseases anddisorders including, for example, diseases or disorders related to agingor stress, diabetes, obesity, neurodegenerative diseases, cardiovasculardisease, blood clotting disorders, inflammation, cancer, and/orflushing, etc. The methods comprise administering to a subject in needthereof a pharmaceutically effective amount of a sirtuin-modulatingcompound, e.g., a sirtuin-activating compound.

While Applicants do not wish to be bound by theory, it is believed thatactivators of the instant invention may interact with a sirtuin at thesame location within the sirtuin protein (e.g., active site or siteaffecting the Km or Vmax of the active site). It is believed that thisis the reason why certain classes of sirtuin activators and inhibitorscan have substantial structural similarity.

In certain embodiments, the sirtuin-modulating compounds describedherein may be taken alone or in combination with other compounds. In oneembodiment, a mixture of two or more sirtuin-modulating compounds may beadministered to a subject in need thereof. In another embodiment, asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein may be administered with one or more of the followingcompounds: resveratrol, butein, fisetin, piceatannol, or quercetin. Inan exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered incombination with nicotinic acid. In another embodiment, asirtuin-modulating compound that decreases the level and/or activity ofa sirtuin protein may be administered with one or more of the followingcompounds: nicotinamide (NAM), suranim; NF023 (a G-protein antagonist);NF279 (a purinergic receptor antagonist); Trolox(6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid);(−)-epigallocatechin (hydroxy on sites 3,5,7,3′,4′,5′);(−)-epigallocatechin gallate (Hydroxy sites 5,7,3′,4′,5′ and gallateester on 3); cyanidin chloride (3,5,7,3′,4′-pentahydroxyflavyliumchloride); delphinidin chloride (3,5,7,3′,4′,5′-hexahydroxyflavyliumchloride); myricetin (cannabiscetin; 3,5,7,3′,4′,5′-hexahydroxyflavone);3,7,3′,4′,5′-pentahydroxyflavone; gossypetin(3,5,7,8,3′,4′-hexahydroxyflavone), sirtinol; and splitomicin. In yetanother embodiment, one or more sirtuin-modulating compounds may beadministered with one or more therapeutic agents for the treatment orprevention of various diseases, including, for example, cancer,diabetes, neurodegenerative diseases, cardiovascular disease, bloodclotting, inflammation, flushing, obesity, ageing, stress, etc. Invarious embodiments, combination therapies comprising asirtuin-modulating compound may refer to (1) pharmaceutical compositionsthat comprise one or more sirtuin-modulating compounds in combinationwith one or more therapeutic agents (e.g., one or more therapeuticagents described herein); and (2) co-administration of one or moresirtuin-modulating compounds with one or more therapeutic agents whereinthe sirtuin-modulating compound and therapeutic agent have not beenformulated in the same compositions (but may be present within the samekit or package, such as a blister pack or other multi-chamber package;connected, separately sealed containers (e.g., foil pouches) that can beseparated by the user; or a kit where the sirtuin modulating compound(s)and other therapeutic agent(s) are in separate vessels). When usingseparate formulations, the sirtuin-modulating compound may beadministered at the same, intermittent, staggered, prior to, subsequentto, or combinations thereof, with the administration of anothertherapeutic agent.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise increasing the protein level of a sirtuin, such as human SIRT1,SIRT2 and/or SIRT3, or homologs thereof. Increasing protein levels canbe achieved by introducing into a cell one or more copies of a nucleicacid that encodes a sirtuin. For example, the level of a sirtuin can beincreased in a mammalian cell by introducing into the mammalian cell anucleic acid encoding the sirtuin, e.g., increasing the level of SIRT1by introducing a nucleic acid encoding the amino acid sequence set forthin GenBank Accession No. NP_(—)036370 and/or increasing the level ofSIRT3 by introducing a nucleic acid encoding the amino acid sequence setforth in GenBank Accession No. AAH01042.

A nucleic acid that is introduced into a cell to increase the proteinlevel of a sirtuin may encode a protein that is at least about 80%, 85%,90%, 95%, 98%, or 99% identical to the sequence of a sirtuin, e.g.,SIRT1 and/or SIRT3 protein. For example, the nucleic acid encoding theprotein may be at least about 80%, 85%, 90%, 95%, 98%, or 99% identicalto a nucleic acid encoding a SIRT1 (e.g. GenBank Accession No.NM_(—)012238) and/or SIRT3 (e.g., GenBank Accession No. BC001042)protein. The nucleic acid may also be a nucleic acid that hybridizes,preferably under stringent hybridization conditions, to a nucleic acidencoding a wild-type sirtuin, e.g., SIRT1 and/or SIRT3 protein.Stringent hybridization conditions may include hybridization and a washin 0.2×SSC at 65° C. When using a nucleic acid that encodes a proteinthat is different from a wild-type sirtuin protein, such as a proteinthat is a fragment of a wild-type sirtuin, the protein is preferablybiologically active, e.g., is capable of deacetylation. It is onlynecessary to express in a cell a portion of the sirtuin that isbiologically active. For example, a protein that differs from wild-typeSIRT1 having GenBank Accession No. NP_(—)036370, preferably contains thecore structure thereof. The core structure sometimes refers to aminoacids 62-293 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 237 to 932 of GenBank Accession No. NM_(—)012238, whichencompasses the NAD binding as well as the substrate binding domains.The core domain of SIRT1 may also refer to about amino acids 261 to 447of GenBank Accession No. NP_(—)036370, which are encoded by nucleotides834 to 1394 of GenBank Accession No. NM_(—)012238; to about amino acids242 to 493 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 777 to 1532 of GenBank Accession No. NM_(—)012238; or toabout amino acids 254 to 495 of GenBank Accession No. NP_(—)036370,which are encoded by nucleotides 813 to 1538 of GenBank Accession No.NM_(—)012238. Whether a protein retains a biological function, e.g.,deacetylation capabilities, can be determined according to methods knownin the art.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise decreasing the protein level of a sirtuin, such as human SIRT1,SIRT2 and/or SIRT3, or homologs thereof. Decreasing a sirtuin proteinlevel can be achieved according to methods known in the art. Forexample, an siRNA, an antisense nucleic acid, or a ribozyme targeted tothe sirtuin can be expressed in the cell. A dominant negative sirtuinmutant, e.g., a mutant that is not capable of deacetylating, may also beused. For example, mutant H363Y of SIRT1, described, e.g., in Luo et al.(2001) Cell 107:137 can be used. Alternatively, agents that inhibittranscription can be used.

Methods for modulating sirtuin protein levels also include methods formodulating the transcription of genes encoding sirtuins, methods forstabilizing/destabilizing the corresponding mRNAs, and other methodsknown in the art.

Aging/Stress

In one embodiment, the invention provides a method extending thelifespan of a cell, extending the proliferative capacity of a cell,slowing aging of a cell, promoting the survival of a cell, delayingcellular senescence in a cell, mimicking the effects of calorierestriction, increasing the resistance of a cell to stress, orpreventing apoptosis of a cell, by contacting the cell with asirtuin-modulating compound of the invention that increases the leveland/or activity of a sirtuin protein. In an exemplary embodiment, themethods comprise contacting the cell with a sirtuin-activating compound.

The methods described herein may be used to increase the amount of timethat cells, particularly primary cells (i.e., cells obtained from anorganism, e.g., a human), may be kept alive in a cell culture. Embryonicstem (ES) cells and pluripotent cells, and cells differentiatedtherefrom, may also be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein to keep thecells, or progeny thereof, in culture for longer periods of time. Suchcells can also be used for transplantation into a subject, e.g., afterex vivo modification.

In one embodiment, cells that are intended to be preserved for longperiods of time may be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. The cells maybe in suspension (e.g., blood cells, serum, biological growth media,etc.) or in tissues or organs. For example, blood collected from anindividual for purposes of transfusion may be treated with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein to preserve the blood cells for longer periods oftime. Additionally, blood to be used for forensic purposes may also bepreserved using a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. Other cells that may be treated toextend their lifespan or protect against apoptosis include cells forconsumption, e.g., cells from non-human mammals (such as meat) or plantcells (such as vegetables).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be applied during developmental and growthphases in mammals, plants, insects or microorganisms, in order to, e.g.,alter, retard or accelerate the developmental and/or growth process.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat cellsuseful for transplantation or cell therapy, including, for example,solid tissue grafts, organ transplants, cell suspensions, stem cells,bone marrow cells, etc. The cells or tissue may be an autograft, anallograft, a syngraft or a xenograft. The cells or tissue may be treatedwith the sirtuin-modulating compound prior toadministration/implantation, concurrently withadministration/implantation, and/or post administration/implantationinto a subject. The cells or tissue may be treated prior to removal ofthe cells from the donor individual, ex vivo after removal of the cellsor tissue from the donor individual, or post implantation into therecipient. For example, the donor or recipient individual may be treatedsystemically with a sirtuin-modulating compound or may have a subset ofcells/tissue treated locally with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. In certainembodiments, the cells or tissue (or donor/recipient individuals) mayadditionally be treated with another therapeutic agent useful forprolonging graft survival, such as, for example, an immunosuppressiveagent, a cytokine, an angiogenic factor, etc.

In yet other embodiments, cells may be treated with a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin proteinin vivo, e.g., to increase their lifespan or prevent apoptosis. Forexample, skin can be protected from aging (e.g., developing wrinkles,loss of elasticity, etc.) by treating skin or epithelial cells with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein. In an exemplary embodiment, skin is contacted with apharmaceutical or cosmetic composition comprising a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.Exemplary skin afflictions or skin conditions that may be treated inaccordance with the methods described herein include disorders ordiseases associated with or caused by inflammation, sun damage ornatural aging. For example, the compositions find utility in theprevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpenfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, psoriasis, skin cancer and the effects of naturalaging. In another embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be used for thetreatment of wounds and/or burns to promote healing, including, forexample, first-, second- or third-degree burns and/or thermal, chemicalor electrical burns. The formulations may be administered topically, tothe skin or mucosal tissue.

Topical formulations comprising one or more sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused as preventive, e.g., chemopreventive, compositions. When used in achemopreventive method, susceptible skin is treated prior to any visiblecondition in a particular individual.

Sirtuin-modulating compounds may be delivered locally or systemically toa subject. In one embodiment, a sirtuin-modulating compound is deliveredlocally to a tissue or organ of a subject by injection, topicalformulation, etc.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used for treating orpreventing a disease or condition induced or exacerbated by cellularsenescence in a subject; methods for decreasing the rate of senescenceof a subject, e.g., after onset of senescence; methods for extending thelifespan of a subject; methods for treating or preventing a disease orcondition relating to lifespan; methods for treating or preventing adisease or condition relating to the proliferative capacity of cells;and methods for treating or preventing a disease or condition resultingfrom cell damage or death. In certain embodiments, the method does notact by decreasing the rate of occurrence of diseases that shorten thelifespan of a subject. In certain embodiments, a method does not act byreducing the lethality caused by a disease, such as cancer.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered to asubject in order to generally increase the lifespan of its cells and toprotect its cells against stress and/or against apoptosis. It isbelieved that treating a subject with a compound described herein issimilar to subjecting the subject to hormesis, i.e., mild stress that isbeneficial to organisms and may extend their lifespan.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to a subject to prevent aging andaging-related consequences or diseases, such as stroke, heart disease,heart failure, arthritis, high blood pressure, and Alzheimer's disease.Other conditions that can be treated include ocular disorders, e.g.,associated with the aging of the eye, such as cataracts, glaucoma, andmacular degeneration. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can also be administered tosubjects for treatment of diseases, e.g., chronic diseases, associatedwith cell death, in order to protect the cells from cell death.Exemplary diseases include those associated with neural cell death,neuronal dysfunction, or muscular cell death or dysfunction, such asParkinson's disease, Alzheimer's disease, multiple sclerosis,amniotropic lateral sclerosis, and muscular dystrophy; AIDS; fulminanthepatitis; diseases linked to degeneration of the brain, such asCreutzfeld-Jakob disease, retinitis pigmentosa and cerebellardegeneration; myelodysplasis such as aplastic anemia; ischemic diseasessuch as myocardial infarction and stroke; hepatic diseases such asalcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such asosteoarthritis; atherosclerosis; alopecia; damage to the skin due to UVlight; lichen planus; atrophy of the skin; cataract; and graftrejections. Cell death can also be caused by surgery, drug therapy,chemical exposure or radiation exposure.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can also be administered to a subject suffering froman acute disease, e.g., damage to an organ or tissue, e.g., a subjectsuffering from stroke or myocardial infarction or a subject sufferingfrom a spinal cord injury. Sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may also be used torepair an alcoholic's liver.

Cardiovascular Disease

In another embodiment, the invention provides a method for treatingand/or preventing a cardiovascular disease by administering to a subjectin need thereof a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein.

Cardiovascular diseases that can be treated or prevented using thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein include cardiomyopathy or myocarditis; such asidiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholiccardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy,and hypertensive cardiomyopathy. Also treatable or preventable usingcompounds and methods described herein are atheromatous disorders of themajor blood vessels (macrovascular disease) such as the aorta, thecoronary arteries, the carotid arteries, the cerebrovascular arteries,the renal arteries, the iliac arteries, the femoral

arteries, and the popliteal arteries. Other vascular diseases that canbe treated or prevented include those related to platelet aggregation,the retinal arterioles, the glomerular arterioles, the vasa nervorum,cardiac arterioles, and associated capillary beds of the eye, thekidney, the heart, and the central and peripheral nervous systems. Thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used for increasing HDL levels in plasmaof an individual.

Yet other disorders that may be treated with sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteininclude restenosis, e.g., following coronary intervention, and disordersrelating to an abnormal level of high density and low densitycholesterol.

In one embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be administered as partof a combination therapeutic with another cardiovascular agent. In oneembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered as part of acombination therapeutic with an anti-arrhythmia agent. In anotherembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered as part of acombination therapeutic with another cardiovascular agent.

Cell Death/Cancer

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to subjects who have recentlyreceived or are likely to receive a dose of radiation or toxin. In oneembodiment, the dose of radiation or toxin is received as part of awork-related or medical procedure, e.g., administered as a prophylacticmeasure. In another embodiment, the radiation or toxin exposure isreceived unintentionally. In such a case, the compound is preferablyadministered as soon as possible after the exposure to inhibit apoptosisand the subsequent development of acute radiation syndrome.

Sirtuin-modulating compounds may also be used for treating and/orpreventing cancer. In certain embodiments, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedfor treating and/or preventing cancer. Calorie restriction has beenlinked to a reduction in the incidence of age-related disordersincluding cancer. Accordingly, an increase in the level and/or activityof a sirtuin protein may be useful for treating and/or preventing theincidence of age-related disorders, such as, for example, cancer.Exemplary cancers that may be treated using a sirtuin-modulatingcompound are those of the brain and kidney; hormone-dependent cancersincluding breast, prostate, testicular, and ovarian cancers; lymphomas,and leukemias. In cancers associated with solid tumors, a modulatingcompound may be administered directly into the tumor. Cancer of bloodcells, e.g., leukemia, can be treated by administering a modulatingcompound into the blood stream or into the bone marrow. Benign cellgrowth, e.g., warts, can also be treated. Other diseases that can betreated include autoimmune diseases, e.g., systemic lupus erythematosus,scleroderma, and arthritis, in which autoimmune cells should be removed.Viral infections such as herpes, HIV, adenovirus, and HTLV-1 associatedmalignant and benign disorders can also be treated by administration ofsirtuin-modulating compound. Alternatively, cells can be obtained from asubject, treated ex vivo to remove certain undesirable cells, e.g.,cancer cells, and administered back to the same or a different subject.

Chemotherapeutic agents may be co-administered with modulating compoundsdescribed herein as having anti-cancer activity, e.g., compounds thatinduce apoptosis, compounds that reduce lifespan or compounds thatrender cells sensitive to stress. Chemotherapeutic agents may be used bythemselves with a sirtuin-modulating compound described herein asinducing cell death or reducing lifespan or increasing sensitivity tostress and/or in combination with other chemotherapeutics agents. Inaddition to conventional chemotherapeutics, the sirtuin-modulatingcompounds described herein may also be used with antisense RNA, RNAi orother polynucleotides to inhibit the expression of the cellularcomponents that contribute to unwanted cellular proliferation.

Combination therapies comprising sirtuin-modulating compounds and aconventional chemotherapeutic agent may be advantageous over combinationtherapies known in the art because the combination allows theconventional chemotherapeutic agent to exert greater effect at lowerdosage. In a preferred embodiment, the effective dose (ED₅₀) for achemotherapeutic agent, or combination of conventional chemotherapeuticagents, when used in combination with a sirtuin-modulating compound isat least 2 fold less than the ED₅₀ for the chemotherapeutic agent alone,and even more preferably at 5 fold, 10 fold or even 25 fold less.Conversely, the therapeutic index (TI) for such chemotherapeutic agentor combination of such chemotherapeutic agent when used in combinationwith a sirtuin-modulating compound described herein can be at least 2fold greater than the TI for conventional chemotherapeutic regimenalone, and even more preferably at 5 fold, 10 fold or even 25 foldgreater.

Neuronal Diseases/Disorders

In certain aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat patientssuffering from neurodegenerative diseases, and traumatic or mechanicalinjury to the central nervous system (CNS), spinal cord or peripheralnervous system (PNS). Neurodegenerative disease typically involvesreductions in the mass and volume of the human brain, which may be dueto the atrophy and/or death of brain cells, which are far more profoundthan those in a healthy person that are attributable to aging.Neurodegenerative diseases can evolve gradually, after a long period ofnormal brain function, due to progressive degeneration (e.g., nerve celldysfunction and death) of specific brain regions. Alternatively,neurodegenerative diseases can have a quick onset, such as thoseassociated with trauma or toxins. The actual onset of brain degenerationmay precede clinical expression by many years. Examples ofneurodegenerative diseases include, but are not limited to, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewybody disease, chorea-acanthocytosis, primary lateral sclerosis, oculardiseases (ocular neuritis), chemotherapy-induced neuropathies (e.g.,from vincristine, paclitaxel, bortezomib), diabetes-induced neuropathiesand Friedreich's ataxia. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can be used to treat thesedisorders and others as described below.

AD is a CNS disorder that results in memory loss, unusual behavior,personality changes, and a decline in thinking abilities. These lossesare related to the death of specific types of brain cells and thebreakdown of connections and their supporting network (e.g. glial cells)between them. The earliest symptoms include loss of recent memory,faulty judgment, and changes in personality. PD is a CNS disorder thatresults in uncontrolled body movements, rigidity, tremor, anddyskinesia, and is associated with the death of brain cells in an areaof the brain that produces dopamine. ALS (motor neuron disease) is a CNSdisorder that attacks the motor neurons, components of the CNS thatconnect the brain to the skeletal muscles.

HD is another neurodegenerative disease that causes uncontrolledmovements, loss of intellectual faculties, and emotional disturbance.Tay-Sachs disease and Sandhoff disease are glycolipid storage diseaseswhere GM2 ganglioside and related glycolipidssubstrates for(3-hexosaminidase accumulate in the nervous system and trigger acuteneurodegeneration.

It is well-known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease, whichcan be treated with sirtuin-modulating compounds of the invention.

Neuronal loss is also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for treating orpreventing neuronal loss due to these prior diseases.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used to treat orprevent any disease or disorder involving axonopathy. Distal axonopathyis a type of peripheral neuropathy that results from some metabolic ortoxic derangement of peripheral nervous system (PNS) neurons. It is themost common response of nerves to metabolic or toxic disturbances, andas such may be caused by metabolic diseases such as diabetes, renalfailure, deficiency syndromes such as malnutrition and alcoholism, orthe effects of toxins or drugs. Those with distal axonopathies usuallypresent with symmetrical glove-stocking sensori-motor disturbances. Deeptendon reflexes and autonomic nervous system (ANS) functions are alsolost or diminished in affected areas.

Diabetic neuropathies are neuropathic disorders that are associated withdiabetes mellitus. Relatively common conditions which may be associatedwith diabetic neuropathy include third nerve palsy; mononeuropathy;mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy;autonomic neuropathy; and thoracoabdominal neuropathy.

Peripheral neuropathy is the medical term for damage to nerves of theperipheral nervous system, which may be caused either by diseases of thenerve or from the side-effects of systemic illness. Major causes ofperipheral neuropathy include seizures, nutritional deficiencies, andHIV, though diabetes is the most likely cause.

In an exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treat orprevent multiple sclerosis (MS), including relapsing MS andmonosymptomatic MS, and other demyelinating conditions, such as, forexample, chromic inflammatory demyelinating polyneuropathy (CIDP), orsymptoms associated therewith.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treattrauma to the nerves, including, trauma due to disease, injury(including surgical intervention), or environmental trauma (e.g.,neurotoxins, alcoholism, etc.).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be useful to prevent, treat, and alleviatesymptoms of various PNS disorders. The term “peripheral neuropathy”encompasses a wide range of disorders in which the nerves outside of thebrain and spinal cord—peripheral nerves—have been damaged. Peripheralneuropathy may also be referred to as peripheral neuritis, or if manynerves are involved, the terms polyneuropathy or polyneuritis may beused.

PNS diseases treatable with sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein include: diabetes,leprosy, Charcot-Marie-Tooth disease, Guillain-Barré syndrome andBrachial Plexus Neuropathies (diseases of the cervical and firstthoracic roots, nerve trunks, cords, and peripheral nerve components ofthe brachial plexus.

In another embodiment, a sirtuin activating compound may be used totreat or prevent a polyglutamine disease. Exemplary polyglutaminediseases include Spinobulbar muscular atrophy (Kennedy disease),Huntington's Disease (HD), Dentatorubral-pallidoluysian atrophy (HawRiver syndrome), Spinocerebellar ataxia type 1, Spinocerebellar ataxiatype 2, Spinocerebellar ataxia type 3 (Machado-Joseph disease),Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, andSpinocerebellar ataxia type 17.

In certain embodiments, the invention provides a method to treat acentral nervous system cell to prevent damage in response to a decreasein blood flow to the cell. Typically the severity of damage that may beprevented will depend in large part on the degree of reduction in bloodflow to the cell and the duration of the reduction. In one embodiment,apoptotic or necrotic cell death may be prevented. In still a furtherembodiment, ischemic-mediated damage, such as cytoxic edema or centralnervous system tissue anoxemia, may be prevented. In each embodiment,the central nervous system cell may be a spinal cell or a brain cell.

Another aspect encompasses administrating a sirtuin activating compoundto a subject to treat a central nervous system ischemic condition. Anumber of central nervous system ischemic conditions may be treated bythe sirtuin activating compounds described herein. In one embodiment,the ischemic condition is a stroke that results in any type of ischemiccentral nervous system damage, such as apoptotic or necrotic cell death,cytoxic edema or central nervous system tissue anoxia. The stroke mayimpact any area of the brain or be caused by any etiology commonly knownto result in the occurrence of a stroke. In one alternative of thisembodiment, the stroke is a brain stem stroke. In another alternative ofthis embodiment, the stroke is a cerebellar stroke. In still anotherembodiment, the stroke is an embolic stroke. In yet another alternative,the stroke may be a hemorrhagic stroke. In a further embodiment, thestroke is a thrombotic stroke.

In yet another aspect, a sirtuin activating compound may be administeredto reduce infarct size of the ischemic core following a central nervoussystem ischemic condition. Moreover, a sirtuin activating compound mayalso be beneficially administered to reduce the size of the ischemicpenumbra or transitional zone following a central nervous systemischemic condition.

In one embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of neurodegenerative disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more anti-neurodegeneration agents.

Blood Coagulation Disorders

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or preventblood coagulation disorders (or hemostatic disorders). As usedinterchangeably herein, the terms “hemostasis”, “blood coagulation,” and“blood clotting” refer to the control of bleeding, including thephysiological properties of vasoconstriction and coagulation. Bloodcoagulation assists in maintaining the integrity of mammaliancirculation after injury, inflammation, disease, congenital defect,dysfunction or other disruption. Further, the formation of blood clotsdoes not only limit bleeding in case of an injury (hemostasis), but maylead to serious organ damage and death in the context of atheroscleroticdiseases by occlusion of an important artery or vein. Thrombosis is thusblood clot formation at the wrong time and place.

Accordingly, the present invention provides anticoagulation andantithrombotic treatments aiming at inhibiting the formation of bloodclots in order to prevent or treat blood coagulation disorders, such asmyocardial infarction, stroke, loss of a limb by peripheral arterydisease or pulmonary embolism.

As used interchangeably herein, “modulating or modulation of hemostasis”and “regulating or regulation of hemostasis” includes the induction(e.g., stimulation or increase) of hemostasis, as well as the inhibition(e.g., reduction or decrease) of hemostasis.

In one aspect, the invention provides a method for reducing orinhibiting hemostasis in a subject by administering a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.The compositions and methods disclosed herein are useful for thetreatment or prevention of thrombotic disorders. As used herein, theterm “thrombotic disorder” includes any disorder or conditioncharacterized by excessive or unwanted coagulation or hemostaticactivity, or a hypercoagulable state. Thrombotic disorders includediseases or disorders involving platelet adhesion and thrombusformation, and may manifest as an increased propensity to formthromboses, e.g., an increased number of thromboses, thrombosis at anearly age, a familial tendency towards thrombosis, and thrombosis atunusual sites.

In another embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of blood coagulation disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinand one or more anti-coagulation or anti-thrombosis agents.

Weight Control

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing weight gain or obesity in a subject. For example,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used, for example, to treat or preventhereditary obesity, dietary obesity, hormone related obesity, obesityrelated to the administration of medication, to reduce the weight of asubject, or to reduce or prevent weight gain in a subject. A subject inneed of such a treatment may be a subject who is obese, likely to becomeobese, overweight, or likely to become overweight. Subjects who arelikely to become obese or overweight can be identified, for example,based on family history, genetics, diet, activity level, medicationintake, or various combinations thereof.

In yet other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered tosubjects suffering from a variety of other diseases and conditions thatmay be treated or prevented by promoting weight loss in the subject.Such diseases include, for example, high blood pressure, hypertension,high blood cholesterol, dyslipidemia, type 2 diabetes, insulinresistance, glucose intolerance, hyperinsulinemia, coronary heartdisease, angina pectoris, congestive heart failure, stroke, gallstones,cholescystitis and cholelithiasis, gout, osteoarthritis, obstructivesleep apnea and respiratory problems, some types of cancer (such asendometrial, breast, prostate, and colon), complications of pregnancy,poor female reproductive health (such as menstrual irregularities,infertility, irregular ovulation), bladder control problems (such asstress incontinence); uric acid nephrolithiasis; psychological disorders(such as depression, eating disorders, distorted body image, and lowself esteem). Finally, patients with AIDS can develop lipodystrophy orinsulin resistance in response to combination therapies for AIDS.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for inhibitingadipogenesis or fat cell differentiation, whether in vitro or in vivo.Such methods may be used for treating or preventing obesity.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for reducingappetite and/or increasing satiety, thereby causing weight loss oravoidance of weight gain. A subject in need of such a treatment may be asubject who is overweight, obese or a subject likely to becomeoverweight or obese. The method may comprise administering daily or,every other day, or once a week, a dose, e.g., in the form of a pill, toa subject. The dose may be an “appetite reducing dose.”

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing weight gain or obesity.For example, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-obesity agents.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered to reducedrug-induced weight gain. For example, a sirtuin-modulating compoundthat increases the level and/or activity of a sirtuin protein may beadministered as a combination therapy with medications that maystimulate appetite or cause weight gain, in particular, weight gain dueto factors other than water retention.

Metabolic Disorders/Diabetes

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing a metabolic disorder, such as insulin-resistance, apre-diabetic state, type II diabetes, and/or complications thereof.Administration of a sirtuin-modulating compounds that increases thelevel and/or activity of a sirtuin protein may increase insulinsensitivity and/or decrease insulin levels in a subject. A subject inneed of such a treatment may be a subject who has insulin resistance orother precursor symptom of type II diabetes, who has type II diabetes,or who is likely to develop any of these conditions. For example, thesubject may be a subject having insulin resistance, e.g., having highcirculating levels of insulin and/or associated conditions, such ashyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucosetolerance, high blood glucose sugar level, other manifestations ofsyndrome X, hypertension, atherosclerosis and lipodystrophy.

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing a metabolic disorder. Forexample, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-diabetic agents.

Inflammatory Diseases

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or prevent adisease or disorder associated with inflammation. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered prior to the onset of, at, or after the initiationof inflammation. When used prophylactically, the compounds arepreferably provided in advance of any inflammatory response or symptom.Administration of the compounds may prevent or attenuate inflammatoryresponses or symptoms.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat orprevent allergies and respiratory conditions, including asthma,bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity,emphysema, chronic bronchitis, acute respiratory distress syndrome, andany chronic obstructive pulmonary disease (COPD). The compounds may beused to treat chronic hepatitis infection, including hepatitis B andhepatitis C.

Additionally, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to treat autoimmunediseases and/or inflammation associated with autoimmune diseases such asarthritis, including rheumatoid arthritis, psoriatic arthritis, andankylosing spondylitis, as well as organ-tissue autoimmune diseases(e.g., Raynaud's syndrome), ulcerative colitis, Crohns Disease, oralmucositis, scleroderma, myasthenia gravis, transplant rejection,endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiplesclerosis, autoimmune thyroiditis, uveitis, systemic lupuserythematosis, Addison's disease, autoimmune polyglandular disease (alsoknown as autoimmune polyglandular syndrome), and Grave's disease.

In certain embodiments, one or more sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein may be takenalone or in combination with other compounds useful for treating orpreventing inflammation.

Flushing

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for reducing theincidence or severity of flushing and/or hot flashes which are symptomsof a disorder. For instance, the subject method includes the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein, alone or in combination with other agents, forreducing incidence or severity of flushing and/or hot flashes in cancerpatients. In other embodiments, the method provides for the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce the incidence or severity of flushing and/orhot flashes in menopausal and post-menopausal woman.

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used as a therapy forreducing the incidence or severity of flushing and/or hot flashes whichare side-effects of another drug therapy, e.g., drug-induced flushing.In certain embodiments, a method for treating and/or preventingdrug-induced flushing comprises administering to a patient in needthereof a formulation comprising at least one flushing inducing compoundand at least one sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. In other embodiments, a method fortreating drug induced flushing comprises separately administering one ormore compounds that induce flushing and one or more sirtuin-modulatingcompounds, e.g., wherein the sirtuin-modulating compound and flushinginducing agent have not been formulated in the same compositions. Whenusing separate formulations, the sirtuin-modulating compound may beadministered (1) at the same as administration of the flushing inducingagent, (2) intermittently with the flushing inducing agent, (3)staggered relative to administration of the flushing inducing agent, (4)prior to administration of the flushing inducing agent, (5) subsequentto administration of the flushing inducing agent, and (6) variouscombination thereof. Exemplary flushing inducing agents include, forexample, niacin, faloxifene, antidepressants, anti-psychotics,chemotherapeutics, calcium channel blockers, and antibiotics.

In one embodiment, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to reduce flushing sideeffects of a vasodilator or an antilipemic agent (includinganticholesteremic agents and lipotropic agents). In an exemplaryembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be used to reduce flushingassociated with the administration of niacin.

In another embodiment, the invention provides a method for treatingand/or preventing hyperlipidemia with reduced flushing side effects. Inanother representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of raloxifene. Inanother representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of antidepressants oranti-psychotic agent. For instance, sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein can be used inconjunction (administered separately or together) with a serotoninreuptake inhibitor, or a 5HT2 receptor antagonist.

In certain embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used as part of atreatment with a serotonin reuptake inhibitor (SRI) to reduce flushing.In still another representative embodiment, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedto reduce flushing side effects of chemotherapeutic agents, such ascyclophosphamide and tamoxifen.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of calcium channel blockers, such as amlodipine.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of antibiotics. For example, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteincan be used in combination with levofloxacin.

Ocular Disorders

One aspect of the present invention is a method for inhibiting, reducingor otherwise treating vision impairment by administering to a patient atherapeutic dosage of sirtuin modulator selected from a compounddisclosed herein, or a pharmaceutically acceptable salt, prodrug or ametabolic derivative thereof.

In certain aspects of the invention, the vision impairment is caused bydamage to the optic nerve or central nervous system. In particularembodiments, optic nerve damage is caused by high intraocular pressure,such as that created by glaucoma. In other particular embodiments, opticnerve damage is caused by swelling of the nerve, which is oftenassociated with an infection or an immune (e.g., autoimmune) responsesuch as in optic neuritis.

In certain aspects of the invention, the vision impairment is caused byretinal damage. In particular embodiments, retinal damage is caused bydisturbances in blood flow to the eye (e.g., arteriosclerosis,vasculitis). In particular embodiments, retinal damage is caused bydisrupton of the macula (e.g., exudative or non-exudative maculardegeneration).

Exemplary retinal diseases include Exudative Age Related MacularDegeneration, Nonexudative Age Related Macular Degeneration, RetinalElectronic Prosthesis and RPE Transplantation Age Related MacularDegeneration, Acute Multifocal Placoid Pigment Epitheliopathy, AcuteRetinal Necrosis, Best Disease, Branch Retinal Artery Occlusion, BranchRetinal Vein Occlusion, Cancer Associated and Related AutoimmuneRetinopathies, Central Retinal Artery Occlusion, Central Retinal VeinOcclusion, Central Serous Chorioretinopathy, Eales Disease, EpimacularMembrane, Lattice Degeneration, Macroaneurysm, Diabetic Macular Edema,Irvine-Gass Macular Edema, Macular Hole, Subretinal NeovascularMembranes, Diffuse Unilateral Subacute Neuroretinitis, NonpseudophakicCystoid Macular Edema, Presumed Ocular Histoplasmosis Syndrome,Exudative Retinal Detachment, Postoperative Retinal Detachment,Proliferative Retinal Detachment, Rhegmatogenous Retinal Detachment,Tractional Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis,Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy,Background Diabetic Retinopathy, Proliferative Diabetic Retinopathy,Hemoglobinopathies Retinopathy, Purtscher Retinopathy, ValsalvaRetinopathy, Juvenile Retinoschisis, Senile Retinoschisis, TersonSyndrome and White Dot Syndromes.

Other exemplary diseases include ocular bacterial infections (e.g.conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viralinfections (e.g. Ocular Herpes Simplex Virus, Varicella Zoster Virus,Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as wellas progressive outer retinal necrosis secondary to HIV or otherHIV-associated and other immunodeficiency-associated ocular diseases. Inaddition, ocular diseases include fungal infections (e.g. Candidachoroiditis, histoplasmosis), protozoal infections (e.g. toxoplasmosis)and others such as ocular toxocariasis and sarcoidosis.

One aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing treatment with achemotherapeutic drug (e.g., a neurotoxic drug, a drug that raisesintraocular pressure such as a steroid), by administering to the subjectin need of such treatment a therapeutic dosage of a sirtuin modulatordisclosed herein.

Another aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing surgery, includingocular or other surgeries performed in the prone position such as spinalcord surgery, by administering to the subject in need of such treatmenta therapeutic dosage of a sirtuin modulator disclosed herein. Ocularsurgeries include cataract, iridotomy and lens replacements.

Another aspect of the invention is the treatment, including inhibitionand prophylactic treatment of age related ocular diseases includecataracts, dry eye, age-related macular degeneration (AMD), retinaldamage and the like, by administering to the subject in need of suchtreatment a therapeutic dosage of a sirtuin modulator disclosed herein.

Another aspect of the invention is the prevention or treatment of damageto the eye caused by stress, chemical insult or radiation, byadministering to the subject in need of such treatment a therapeuticdosage of a sirtuin modulator disclosed herein. Radiation orelectromagnetic damage to the eye can include that caused by CRT's orexposure to sunlight or UV.

In one embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of ocular disorders orsecondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more therapeutic agents for the treatment of an ocular disorder.

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for reducing intraocular pressure. In anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing glaucoma. In yet anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing optic neuritis. In oneembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing CMV Retinopathy. In anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing multiple sclerosis.

Mitochondrial-Associated Diseases and Disorders

In certain embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity. The methods involve administering to a subject in need thereofa therapeutically effective amount of a sirtuin activating compound.Increased mitochondrial activity refers to increasing activity of themitochondria while maintaining the overall numbers of mitochondria(e.g., mitochondrial mass), increasing the numbers of mitochondriathereby increasing mitochondrial activity (e.g., by stimulatingmitochondrial biogenesis), or combinations thereof. In certainembodiments, diseases and disorders that would benefit from increasedmitochondrial activity include diseases or disorders associated withmitochondrial dysfunction.

In certain embodiments, methods for treating diseases or disorders thatwould benefit from increased mitochondrial activity may compriseidentifying a subject suffering from a mitochondrial dysfunction.Methods for diagnosing a mitochondrial dysfunction may involve moleculargenetic, pathologic and/or biochemical analyses. Diseases and disordersassociated with mitochondrial dysfunction include diseases and disordersin which deficits in mitochondrial respiratory chain activity contributeto the development of pathophysiology of such diseases or disorders in amammal. Diseases or disorders that would benefit from increasedmitochondrial activity generally include for example, diseases in whichfree radical mediated oxidative injury leads to tissue degeneration,diseases in which cells inappropriately undergo apoptosis, and diseasesin which cells fail to undergo apoptosis.

In certain embodiments, the invention provides methods for treating adisease or disorder that would benefit from increased mitochondrialactivity that involves administering to a subject in need thereof one ormore sirtuin activating compounds in combination with anothertherapeutic agent such as, for example, an agent useful for treatingmitochondrial dysfunction or an agent useful for reducing a symptomassociated with a disease or disorder involving mitochondrialdysfunction.

In exemplary embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity by administering to a subject a therapeutically effectiveamount of a sirtuin activating compound. Exemplary diseases or disordersinclude, for example, neuromuscular disorders (e.g., Friedreich'sAtaxia, muscular dystrophy, multiple sclerosis, etc.), disorders ofneuronal instability (e.g., seizure disorders, migrane, etc.),developmental delay, neurodegenerative disorders (e.g., Alzheimer'sDisease, Parkinson's Disease, amyotrophic lateral sclerosis, etc.),ischemia, renal tubular acidosis, age-related neurodegeneration andcognitive decline, chemotherapy fatigue, age-related orchemotherapy-induced menopause or irregularities of menstrual cycling orovulation, mitochondrial myopathies, mitochondrial damage (e.g., calciumaccumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), andmitochondrial deregulation.

Muscular dystrophy refers to a family of diseases involvingdeterioration of neuromuscular structure and function, often resultingin atrophy of skeletal muscle and myocardial dysfunction, such asDuchenne muscular dystrophy. In certain embodiments, sirtuin activatingcompounds may be used for reducing the rate of decline in muscularfunctional capacities and for improving muscular functional status inpatients with muscular dystrophy.

In certain embodiments, sirtuin modulating compounds may be useful fortreatment mitochondrial myopathies. Mitochondrial myopathies range frommild, slowly progressive weakness of the extraocular muscles to severe,fatal infantile myopathies and multisystem encephalomyopathies. Somesyndromes have been defined, with some overlap between them. Establishedsyndromes affecting muscle include progressive external ophthalmoplegia,the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy,cardiac conduction defects, cerebellar ataxia, and sensorineuraldeafness), the MELAS syndrome (mitochondrial encephalomyopathy, lacticacidosis, and stroke-like episodes), the MERFF syndrome (myoclonicepilepsy and ragged red fibers), limb-girdle distribution weakness, andinfantile myopathy (benign or severe and fatal).

In certain embodiments, sirtuin activating compounds may be useful fortreating patients suffering from toxic damage to mitochondria, such as,toxic damage due to calcium accumulation, excitotoxicity, nitric oxideexposure, drug induced toxic damage, or hypoxia.

In certain embodiments, sirtuin activating compounds may be useful fortreating diseases or disorders associated with mitochondrialderegulation.

Muscle Performance

In other embodiments, the invention provides methods for enhancingmuscle performance by administering a therapeutically effective amountof a sirtuin activating compound. For example, sirtuin activatingcompounds may be useful for improving physical endurance (e.g., abilityto perform a physical task such as exercise, physical labor, sportsactivities, etc.), inhibiting or retarding physical fatigues, enhancingblood oxygen levels, enhancing energy in healthy individuals, enhanceworking capacity and endurance, reducing muscle fatigue, reducingstress, enhancing cardiac and cardiovascular function, improving sexualability, increasing muscle ATP levels, and/or reducing lactic acid inblood. In certain embodiments, the methods involve administering anamount of a sirtuin activating compound that increase mitochondrialactivity, increase mitochondrial biogenesis, and/or increasemitochondrial mass.

Sports performance refers to the ability of the athlete's muscles toperform when participating in sports activities. Enhanced sportsperformance, strength, speed and endurance are measured by an increasein muscular contraction strength, increase in amplitude of musclecontraction, shortening of muscle reaction time between stimulation andcontraction. Athlete refers to an individual who participates in sportsat any level and who seeks to achieve an improved level of strength,speed and endurance in their performance, such as, for example, bodybuilders, bicyclists, long distance runners, short distance runners,etc. Enhanced sports performance in manifested by the ability toovercome muscle fatigue, ability to maintain activity for longer periodsof time, and have a more effective workout.

In the arena of athlete muscle performance, it is desirable to createconditions that permit competition or training at higher levels ofresistance for a prolonged period of time.

It is contemplated that the methods of the present invention will alsobe effective in the treatment of muscle related pathological conditions,including acute sarcopenia, for example, muscle atrophy and/or cachexiaassociated with burns, bed rest, limb immobilization, or major thoracic,abdominal, and/or orthopedic surgery.

In certain embodiments, the invention provides novel dietarycompositions comprising sirtuin modulators, a method for theirpreparation, and a method of using the compositions for improvement ofsports performance. Accordingly, provided are therapeutic compositions,foods and beverages that have actions of improving physical enduranceand/or inhibiting physical fatigues for those people involved inbroadly-defined exercises including sports requiring endurance andlabors requiring repeated muscle exertions. Such dietary compositionsmay additional comprise electrolytes, caffeine, vitamins, carbohydrates,etc.

Other Uses

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used for treating or preventing viralinfections (such as infections by influenza, herpes or papilloma virus)or as antifungal agents. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered as part of a combination drug therapy with anothertherapeutic agent for the treatment of viral diseases. In anotherembodiment, sirtuin-modulating compounds that increase the level and/oractivity of a sirtuin protein may be administered as part of acombination drug therapy with another anti-fungal agent.

Subjects that may be treated as described herein include eukaryotes,such as mammals, e.g., humans, ovines, bovines, equines, porcines,canines, felines, non-human primate, mice, and rats. Cells that may betreated include eukaryotic cells, e.g., from a subject described above,or plant cells, yeast cells and prokaryotic cells, e.g., bacterialcells. For example, modulating compounds may be administered to farmanimals to improve their ability to withstand farming conditions longer.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance, and resistance to apoptosis in plants. In one embodiment, acompound is applied to plants, e.g., on a periodic basis, or to fungi.In another embodiment, plants are genetically modified to produce acompound. In another embodiment, plants and fruits are treated with acompound prior to picking and shipping to increase resistance to damageduring shipping. Plant seeds may also be contacted with compoundsdescribed herein, e.g., to preserve them.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for modulatinglifespan in yeast cells. Situations in which it may be desirable toextend the lifespan of yeast cells include any process in which yeast isused, e.g., the making of beer, yogurt, and bakery items, e.g., bread.Use of yeast having an extended lifespan can result in using less yeastor in having the yeast be active for longer periods of time. Yeast orother mammalian cells used for recombinantly producing proteins may alsobe treated as described herein.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance and resistance to apoptosis in insects. In this embodiment,compounds would be applied to useful insects, e.g., bees and otherinsects that are involved in pollination of plants. In a specificembodiment, a compound would be applied to bees involved in theproduction of honey. Generally, the methods described herein may beapplied to any organism, e.g., eukaryote, that may have commercialimportance. For example, they can be applied to fish (aquaculture) andbirds (e.g., chicken and fowl).

Higher doses of sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may also be used as a pesticide byinterfering with the regulation of silenced genes and the regulation ofapoptosis during development. In this embodiment, a compound may beapplied to plants using a method known in the art that ensures thecompound is bio-available to insect larvae, and not to plants.

At least in view of the link between reproduction and longevity,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can be applied to affect the reproduction of organismssuch as insects, animals and microorganisms.

4. Assays

Yet other methods contemplated herein include screening methods foridentifying compounds or agents that modulate sirtuins. An agent may bea nucleic acid, such as an aptamer. Assays may be conducted in a cellbased or cell free format. For example, an assay may comprise incubating(or contacting) a sirtuin with a test agent under conditions in which asirtuin can be modulated by an agent known to modulate the sirtuin, andmonitoring or determining the level of modulation of the sirtuin in thepresence of the test agent relative to the absence of the test agent.The level of modulation of a sirtuin can be determined by determiningits ability to deacetylate a substrate. Exemplary substrates areacetylated peptides which can be obtained from BIOMOL (Plymouth Meeting,Pa.). Preferred substrates include peptides of p53, such as thosecomprising an acetylated K382. A particularly preferred substrate is theFluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptideArg-His-Lys-Lys. Other substrates are peptides from human histones H3and H4 or an acetylated amino acid. Substrates may be fluorogenic. Thesirtuin may be SIRT1, Sir2, SIRT3, or a portion thereof. For example,recombinant SIRT1 can be obtained from BIOMOL. The reaction may beconducted for about 30 minutes and stopped, e.g., with nicotinamide. TheHDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOLResearch Laboratories) may be used to determine the level ofacetylation. Similar assays are described in Bitterman et al. (2002) J.Biol. Chem. 277:45099. The level of modulation of the sirtuin in anassay may be compared to the level of modulation of the sirtuin in thepresence of one or more (separately or simultaneously) compoundsdescribed herein, which may serve as positive or negative controls.Sirtuins for use in the assays may be full length sirtuin proteins orportions thereof. Since it has been shown herein that activatingcompounds appear to interact with the N-terminus of SIRT1, proteins foruse in the assays include N-terminal portions of sirtuins, e.g., aboutamino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or 1-252 ofSir2.

In one embodiment, a screening assay comprises (i) contacting a sirtuinwith a test agent and an acetylated substrate under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test agent; and (ii) determining the level of acetylation of thesubstrate, wherein a lower level of acetylation of the substrate in thepresence of the test agent relative to the absence of the test agentindicates that the test agent stimulates deacetylation by the sirtuin,whereas a higher level of acetylation of the substrate in the presenceof the test agent relative to the absence of the test agent indicatesthat the test agent inhibits deacetylation by the sirtuin.

Methods for identifying an agent that modulates, e.g., stimulates,sirtuins in vivo may comprise (i) contacting a cell with a test agentand a substrate that is capable of entering a cell in the presence of aninhibitor of class I and class II HDACs under conditions appropriate forthe sirtuin to deacetylate the substrate in the absence of the testagent; and (ii) determining the level of acetylation of the substrate,wherein a lower level of acetylation of the substrate in the presence ofthe test agent relative to the absence of the test agent indicates thatthe test agent stimulates deacetylation by the sirtuin, whereas a higherlevel of acetylation of the substrate in the presence of the test agentrelative to the absence of the test agent indicates that the test agentinhibits deacetylation by the sirtuin. A preferred substrate is anacetylated peptide, which is also preferably fluorogenic, as furtherdescribed herein. The method may further comprise lysing the cells todetermine the level of acetylation of the substrate. Substrates may beadded to cells at a concentration ranging from about 1 μM to about 10mM, preferably from about 10 μM to 1 mM, even more preferably from about100 μM to 1 mM, such as about 200 μM. A preferred substrate is anacetylated lysine, e.g., E-acetyl lysine (Fluor de Lys, FdL) or Fluor deLys-SIRT1. A preferred inhibitor of class I and class II HDACs istrichostatin A (TSA), which may be used at concentrations ranging fromabout 0.01 to 100 μM, preferably from about 0.1 to 10 μM, such as 1 μM.Incubation of cells with the test compound and the substrate may beconducted for about 10 minutes to 5 hours, preferably for about 1-3hours. Since TSA inhibits all class I and class II HDACs, and thatcertain substrates, e.g., Fluor de Lys, is a poor substrate for SIRT2and even less a substrate for SIRT3-7, such an assay may be used toidentify modulators of SIRT1 in vivo.

5. Pharmaceutical Compositions

The sirtuin-modulating compounds described herein may be formulated in aconventional manner using one or more physiologically orpharmaceutically acceptable carriers or excipients. For example,sirtuin-modulating compounds and their pharmaceutically acceptable saltsand solvates may be formulated for administration by, for example,injection (e.g. SubQ, IM, IP), inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, sublingual, transdermal,nasal, parenteral or rectal administration. In one embodiment, asirtuin-modulating compound may be administered locally, at the sitewhere the target cells are present, i.e., in a specific tissue, organ,or fluid (e.g., blood, cerebrospinal fluid, etc.).

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a compound of Structural Formula (III), asdefined above, or a compound having the Structural Formula (VI), whereineither:

i. X is —C(O)—NH—CR¹⁵R¹⁶-†; and

-   -   each of Z¹¹, Z¹², Z¹³, Z¹⁴ and Y is CR or

ii. X is —C(O)—NH—CR¹⁵R¹⁶-†; and

-   -   R¹¹ and R¹² are each optionally substituted aryl; or

iii. X is —NH—C(O)-†; and

-   -   R¹² is bicyclic heterocycle; and        a pharmaceutically acceptable carrier.

Sirtuin-modulating compounds can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For parenteral administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets, lozenges, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For administration by inhalation (e.g., pulmonary delivery),sirtuin-modulating compounds may be conveniently delivered in the formof an aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Sirtuin-modulating compounds may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Sirtuin-modulating compounds may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, sirtuin-modulatingcompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, sirtuin-modulating compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Controlledrelease formula also includes patches.

In certain embodiments, the compounds described herein can be formulatedfor delivery to the central nervous system (CNS) (reviewed in Begley,Pharmacology & Therapeutics 104: 29-45 (2004)). Conventional approachesfor drug delivery to the CNS include: neurosurgical strategies (e.g.,intracerebral injection or intracerebroventricular infusion); molecularmanipulation of the agent (e.g., production of a chimeric fusion proteinthat comprises a transport peptide that has an affinity for anendothelial cell surface molecule in combination with an agent that isitself incapable of crossing the BBB) in an attempt to exploit one ofthe endogenous transport pathways of the BBB; pharmacological strategiesdesigned to increase the lipid solubility of an agent (e.g., conjugationof water-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide).

Liposomes are a further drug delivery system which is easily injectable.Accordingly, in the method of invention the active compounds can also beadministered in the form of a liposome delivery system. Liposomes arewell-known by a person skilled in the art. Liposomes can be formed froma variety of phospholipids, such as cholesterol, stearylamine ofphosphatidylcholines. Liposomes being usable for the method of inventionencompass all types of liposomes including, but not limited to, smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles.

Another way to produce a formulation, particularly a solution, of asirtuin modulator such as resveratrol or a derivative thereof, isthrough the use of cyclodextrin. By cyclodextrin is meant α-, β-, orγ-cyclodextrin. Cyclodextrins are described in detail in Pitha et al.,U.S. Pat. No. 4,727,064, which is incorporated herein by reference.Cyclodextrins are cyclic oligomers of glucose; these compounds forminclusion complexes with any drug whose molecule can fit into thelipophile-seeking cavities of the cyclodextrin molecule.

Rapidly disintegrating or dissolving dosage forms are useful for therapid absorption, particularly buccal and sublingual absorption, ofpharmaceutically active agents. Fast melt dosage forms are beneficial topatients, such as aged and pediatric patients, who have difficulty inswallowing typical solid dosage forms, such as caplets and tablets.Additionally, fast melt dosage forms circumvent drawbacks associatedwith, for example, chewable dosage forms, wherein the length of time anactive agent remains in a patient's mouth plays an important role indetermining the amount of taste masking and the extent to which apatient may object to throat grittiness of the active agent.

Pharmaceutical compositions (including cosmetic preparations) maycomprise from about 0.00001 to 100% such as from 0.001 to 10% or from0.1% to 5% by weight of one or more sirtuin-modulating compoundsdescribed herein. In another embodiment, the pharmaceutical compositioncomprises: (i) 0.05 to 1000 mg of the compounds of the invention, or apharmaceutically acceptable salt thereof, and (ii) 0.1 to 2 grams of oneor more pharmaceutically acceptable excipients.

In one embodiment, a sirtuin-modulating compound described herein, isincorporated into a topical formulation containing a topical carrierthat is generally suited to topical drug administration and comprisingany such material known in the art. The topical carrier may be selectedso as to provide the composition in the desired form, e.g., as anointment, lotion, cream, microemulsion, gel, oil, solution, or the like,and may be comprised of a material of either naturally occurring orsynthetic origin. It is preferable that the selected carrier notadversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like.

Formulations may be colorless, odorless ointments, lotions, creams,microemulsions and gels.

Sirtuin-modulating compounds may be incorporated into ointments, whichgenerally are semisolid preparations which are typically based onpetrolatum or other petroleum derivatives. The specific ointment base tobe used, as will be appreciated by those skilled in the art, is one thatwill provide for optimum drug delivery, and, preferably, will providefor other desired characteristics as well, e.g., emolliency or the like.As with other carriers or vehicles, an ointment base should be inert,stable, nonirritating and nonsensitizing.

Sirtuin-modulating compounds may be incorporated into lotions, whichgenerally are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and maycomprise a liquid oily emulsion of the oil-in-water type.

Sirtuin-modulating compounds may be incorporated into creams, whichgenerally are viscous liquid or semisolid emulsions, either oil-in-wateror water-in-oil. Cream bases are water-washable, and contain an oilphase, an emulsifier and an aqueous phase. The oil phase is generallycomprised of petrolatum and a fatty alcohol such as cetyl or stearylalcohol; the aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation, as explained in Remington's, supra,is generally a nonionic, anionic, cationic or amphoteric surfactant.

Sirtuin-modulating compounds may be incorporated into microemulsions,which generally are thermodynamically stable, isotropically cleardispersions of two immiscible liquids, such as oil and water, stabilizedby an interfacial film of surfactant molecules (Encyclopedia ofPharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9).

Sirtuin-modulating compounds may be incorporated into gel formulations,which generally are semisolid systems consisting of either suspensionsmade up of small inorganic particles (two-phase systems) or largeorganic molecules distributed substantially uniformly throughout acarrier liquid (single phase gels). Although gels commonly employaqueous carrier liquid, alcohols and oils can be used as the carrierliquid as well.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In certain topical formulations, the active agent is present in anamount in the range of approximately 0.25 wt. % to 75 wt. % of theformulation, preferably in the range of approximately 0.25 wt. % to 30wt. % of the formulation, more preferably in the range of approximately0.5 wt. % to 15 wt. % of the formulation, and most preferably in therange of approximately 1.0 wt. % to 10 wt. % of the formulation.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a sirtuin-modulating compound, or byinsertion of a sustained release device that releases asirtuin-modulating compound. A sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein may bedelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the cornealand internal regions of the eye, as for example the anterior chamber,posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically-acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour. In a further alternative, the compounds may beadministered systemically, such as by intravenous infusion or injection,for treatment of the eye.

Sirtuin-modulating compounds described herein may be stored in oxygenfree environment. For example, resveratrol or analog thereof can beprepared in an airtight capsule for oral administration, such asCapsugel from Pfizer, Inc.

Cells, e.g., treated ex vivo with a sirtuin-modulating compound, can beadministered according to methods for administering a graft to asubject, which may be accompanied, e.g., by administration of animmunosuppressant drug, e.g., cyclosporin A. For general principles inmedicinal formulation, the reader is referred to Cell Therapy: Stem CellTransplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn& W. Sheridan eds, Cambridge University Press, 1996; and HematopoieticStem Cell Therapy, E. D. Ball, J. Lister & p. Law, ChurchillLivingstone, 2000.

Toxicity and therapeutic efficacy of sirtuin-modulating compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. The LD₅₀ is the dose lethal to 50% of thepopulation. The ED₅₀ is the dose therapeutically effective in 50% of thepopulation. The dose ratio between toxic and therapeutic effects(LD₅₀/ED₅₀) is the therapeutic index. Sirtuin-modulating compounds thatexhibit large therapeutic indexes are preferred. Whilesirtuin-modulating compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

6. Kits

Also provided herein are kits, e.g., kits for therapeutic purposes orkits for modulating the lifespan of cells or modulating apoptosis. A kitmay comprise one or more sirtuin-modulating compounds, e.g., inpremeasured doses. A kit may optionally comprise devices for contactingcells with the compounds and instructions for use. Devices includesyringes, stents and other devices for introducing a sirtuin-modulatingcompound into a subject (e.g., the blood vessel of a subject) orapplying it to the skin of a subject.

In yet another embodiment, the invention provides a composition ofmatter comprising a sirtruin modulator of this invention and anothertherapeutic agent (the same ones used in combination therapies andcombination compositions) in separate dosage forms, but associated withone another. The term “associated with one another” as used herein meansthat the separate dosage forms are packaged together or otherwiseattached to one another such that it is readily apparent that theseparate dosage forms are intended to be sold and administered as partof the same regimen. The agent and the sirtruin modulator are preferablypackaged together in a blister pack or other multi-chamber package, oras connected, separately sealed containers (such as foil pouches or thelike) that can be separated by the user (e.g., by tearing on score linesbetween the two containers).

In still another embodiment, the invention provides a kit comprising inseparate vessels, a) a sirtruin modulator of this invention; and b)another another therapeutic agent such as those described elsewhere inthe specification.

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. p. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Preparation of 8-nitro-2-(3-(trifluoromethyl)phenyl)quinoline

3-Trifluoromethylbenzaldehyde (20.0 g, 0.115 mol) was taken up in 500 mLof CH₃CN along with (triphenylphosphoranylidene)acetaldehyde (35 g,0.115 mol). The reaction mixture was stirred at room temperature for 18h. It was then concentrated under reduced pressure. The resultingresidue was taken up in 800 mL of 1:1 pentane/EtOAc and filtered. Thefiltrate was concentrated under reduced pressure to afford crude(E)-3-(3-(trifluoromethyl)phenyl)acrylaldehyde as a dark red oil. Thismaterial was taken up in 30 mL of CH₂Cl₂. The resulting mixture wasslowly added to a suspension of 2-nitroaniline (4 g, 0.029 mol) inconcentrated HCl (50 mL) at 90° C. over a period of 30 min. Theresulting reaction mixture was stirred at 90° C. for an additional 1 h.The reaction mixture was cooled to room temperature and washed withCH₂Cl₂ (2×100 mL). The aqueous layer was neutralized with 5% aqueousNaOH and extracted with CH₂Cl₂. The combined organic layers were dried(Na₂SO₄) and concentrated under reduced pressure. The resulting residuewas purified by chromatography (pentane/EtOAc) to afford 800 mg of8-nitro-2-(3-(trifluoromethyl)phenyl)quinoline. MS (ESI) calcd forC₁₆H₉F₃N₂O₂ (m/z): 318.06, found: 319 [M+1].

The following material was prepared in a similar fashion:

-   a. 8-nitro-2-(3-(trifluoromethoxy)phenyl)quinoline

Preparation of N-(2-phenylquinolin-8-yl)pyrazine-2-carboxamide

P Step 1) Preparation of 2-phenylquinolin-8-amine

8-Nitro-2-phenylquinoline was prepared according to the procedureoutlined by Elderfield et al in J. American Chemical Society (1946), vol68, p. 1589. In a typical run, 8-nitro-2-phenylquinoline (510 mg) wasdissolved in 100 mL of MeOH. After the addition of 10% Pd on C (50 mg),the reaction mixture was thoroughly purged with nitrogen. It was thenstirred vigorously at room temperature under 1 atm of hydrogen for 18 h.The reaction mixture was filtered through Celite and the filtrate wasconcentrated under reduced pressure to afford 380 mg of2-phenylquinolin-8-amine. MS (ESI) calcd for C₁₅H₁₂N₂ (m/z): 220.10,found: 221 [M+1].

The following materials were prepared in a similar fashion:

-   a. 2-(3-(trifluoromethyl)phenyl)quinolin-8-amine-   b. 2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine

Step 2) Preparation of N-(2-phenylquinolin-8-yl)pyrazine-2-carboxamide

2-Phenylquinolin-8-amine (60 mg, 0.27 mmol) was taken up in 2 mL of DMFalong with pyrazine-2-carboxylic acid (34 mg, 0.27 mmol), HATU (207 mg,0.54 mmol) and DIEA (95 μL, 0.54 mmol). The reaction mixture was stirredat room temperature for 18 h. It was then diluted with EtOAc and washedwith water. The organic layer was dried (Na₂SO₄) and concentrated underreduced pressure. The resulting residue was purified by preparative HPLC(using aqueous CH₃CN that has been buffered with 0.1% TFA) to afford 10mg of the product. MS (ESI) calcd for C₂₀H₁₄N₄O (m/z): 326.12, found:327 [M+1].

The following materials were prepared in a similar fashion:

-   a. N-(2-phenylquinolin-8-yl)-3-(pyrrolidin-1-yl)benzamide-   b. N-(2-phenylquinolin-8-yl)thiazole-4-carboxamide-   c. N-(2-phenylquinolin-8-yl)-3-(trifluoromethoxy)benzamide-   d.    N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)pyrazine-2-carboxamide-   e.    N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   f.    N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)pyrazine-2-carboxamide-   g.    N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   h. N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)benzamide-   i. 2-fluoro-N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)benzamide-   j. 3-fluoro-N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)benzamide-   k. 4-fluoro-N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)benzamide-   l.    N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)quinoxaline-2-carboxamide-   m.    N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)oxazole-4-carboxamide-   n.    N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)thiophene-2-carboxamide-   o. 3-methoxy-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide-   p.    2-phenyl-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   q.    3-(dimethylamino)-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide-   r.    3-((4-methylpiperazin-1-yl)methyl)-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide-   s.    N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)thiazole-5-carboxamide-   t.    1-methyl-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)-1H-imidazole-4-carboxamide-   u.    1-methyl-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)-1H-imidazole-2-carboxamide-   v.    N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)-1H-pyrazole-3-carboxamide-   w.    1-methyl-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)-1H-pyrazole-3-carboxamide-   x.    3-(2-morpholinoethoxy)-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide

Preparation of (R)-6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)picolinicacid

Ethyl 6-hydroxypicolinate (500 mg, 2.7 mmol),(R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1.11 mL, 3.0 eq) and NaH(60% dispersion in mineral oil, 385 mg, 3.3 eq) in THF was refluxed 18hours. The reaction mixture was cooled to room temperature, acidified topH=4, added to brine and extracted with ethyl acetate. The organic layerwas dried, concentrated and recrystallized from pentane/ethylacetate toobtain (R)-6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)picolinic acid(500 mg, 74% yield).

Preparation of (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoicacid

Step 1) Preparation of (R)-methyl3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoate

A mixture of methyl 3-hydroxybenzoate (10.0 g, 65.8 mmol),(S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane (13.0 g, 98.7 mmol) andK₂CO₃ (18.0 g, 132 mmol) in DMF (100 ml) was stirred for 18 h at 160° C.The mixture was diluted with water (150 mL) and adjusted to pH=6 by theaddition of 3N HCl. The mixture was extracted with ethyl acetate (200ml×3) and the combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure. The residue was purified by columnchromatography (10% Ethyl acetate in petroleum ether) to give (R)-methyl3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoate as a brown oil, (8.5g, 49% yield).

Step 2) Preparation of(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoic acid

To a solution of (R)-methyl3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoate (8.5 g, 32 mmol) inTHF (80 ml) was added a solution of LiOH (2.3 g, 96 mmol) in water (20ml). The mixture was stirred for 15 hours at 40° C. The mixture wasconcentrated and diluted with saturated solution of Na₂CO₃ (50 ml),washed with ethyl acetate (50 ml×2), The aqueous layer was adjusted topH=4 by addition of aqueous 3N HCl. The precipitate was collected byfiltration and the filtered cake was dried in vacuo to obtain(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoic acid as a whitesolid, (5.8 g, 72% yield).

The following material was prepared in a similar fashion:

-   a. (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzoic acid

Preparation of N-(2-phenylquinolin-8-yl)piperidine-4-carboxamide

2-Phenylquinolin-8-amine (60 mg, 0.27 mmol) was subjected to the samegeneral amide coupling procedure outlined above using1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid. The resultingintermediate, namely tert-butyl4-(2-phenylquinolin-8-ylcarbamoyl)piperidine-1-carboxylate, was furthertreated with 2 mL of 25% TFA in CH₂Cl₂ for 6 h. The reaction mixture wasconcentrated under reduced pressure. The resulting residue was purifiedby preparative HPLC (using aqueous CH₃CN that has been buffered with0.1% TFA) to afford 25 mg of the product. MS (ESI) calcd for C₂₁H₂₁N₃O(m/z): 331.17, found: 332 [M+1].

Preparation of N-(2-(3-fluorophenyl)quinolin-8-yl)thiazole-4-carboxamide

2-Chloro-8-nitroquinoline was prepared according to the procedureoutlined by Kimber et al in Aust J. Chem. (2003), vol 56, pgs. 39-44.

Step 1) Preparation of 2-chloroquinolin-8-amine

A solution of 2-chloro-8-nitroquinoline (1.02 grams), iron powder (2.05grams) and NH₄Cl (2.6 grams) in 5:1 EtOH:Water (50 mL) was refluxed for9 hours. After the reaction was complete, the solution was cooled to 60°C. and filtered through celite. The cake was washed with isopropylalcohol followed by ethyl acetate. The filtrate was concentrated todryness, dissolved in ethyl acetate and washed with water, diluteaqueous NaHCO₃, brine and dried (Na₂SO₄) and concentrated to an oil. Thedesired product crystallized with the addition of pentane, brown solid(0.818 grams).

Step 2) Preparation of N-(2-chloroquinolin-8-yl)thiazole-4-carboxamide

A solution of 2-chloroquinolin-8-amine (222 mg), thiazole-4-carboxylicacid (129 mg, 1 eq), HATU (570 mg, 1.5 eq), and DIEA (246 uL, 2.0 eq) inDMF (3 mL) was stirred at room temperature overnight. The product wasprecipitated by the addition of water (20 mL), the product was collectedby filtration and recrystallized from methanol to obtain the product asa grey solid (194 mg).

The following material was prepared in a similar fashion:

-   a. 2-chloro-N-(pyrazin-2-yl)quinoline-8-carboxamide

Step 3) Preparation ofN-(2-(3-fluorophenyl)quinolin-8-yl)thiazole-4-carboxamide

A solution of N-(2-chloroquinolin-8-yl)thiazole-4-carboxamide (29 mg,0.100 mmol), 3-fluorophenylboronic acid (28 mg, 2 eq.), CsCO₃ (65 mg, 2eq.), Pd(dppf)Cl₂.DCM (4 mg, 0.05 eq.) in DME (2 mL) was microwaveheated (140° C.×15 min.). The reaction was filtered and concentrated.The residue was diluted with ethyl acetate, washed with saturatedaqueous NaHCO₃, dried (Na₂SO₄) and concentrated. The product waspurified by column chromatography (0 to 100% EtOAc in Pentane) to obtain8.6 mg of N-(2-(3-fluorophenyl)quinolin-8-yl)thiazole-4-carboxamide. MS(ESI) calcd for C₁₉H₁₃FN₃OS (m/z): 350.08, found: 350.1 [M+1].

The following materials were prepared in a similar fashion:

-   a. N-(2-(2-fluorophenyl)quinolin-8-yl)thiazole-4-carboxamide-   b. N-(2-(pyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   c. N-(2-(pyridin-4-yl)quinolin-8-yl)thiazole-4-carboxamide-   d. N-(2-(3,5-difluorophenyl)quinolin-8-yl)thiazole-4-carboxamide-   e. N-(2-m-tolylquinolin-8-yl)thiazole-4-carboxamide-   f. N-(2-(3-cyanophenyl)quinolin-8-yl)thiazole-4-carboxamide-   g.    N-(2-(3-(methylsulfonyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   h.    N-(2-(2-(trifluoromethoxy)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   i.    N-(2-(2-(methylsulfonyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   j.    N-(2-(4-(methylsulfonyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   k.    N-(2-(benzo[d][1,3]dioxol-5-yl)quinolin-8-yl)thiazole-4-carboxamide-   l. N-(2-(3-formylphenyl)quinolin-8-yl)thiazole-4-carboxamide-   m. N-(2-(pyridin-3-yl)quinolin-8-yl)pyrazine-2-carboxamide-   n. N-(2-(6-fluoropyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   o. N-(2-(2-hydroxyphenyl)quinolin-8-yl)thiazole-4-carboxamide-   p. N-(2-(3-hydroxyphenyl)quinolin-8-yl)thiazole-4-carboxamide-   q. N-(2-(4-hydroxyphenyl)quinolin-8-yl)thiazole-4-carboxamide-   r. N-(2-(2-methylpyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   s. N-(2-(2-methylpyridin-4-yl)quinolin-8-yl)thiazole-4-carboxamide-   t. N-(2-(6-methylpyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   u. N-(2-(5-fluoropyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   v. N-(2-(3-morpholinophenyl)quinolin-8-yl)thiazole-4-carboxamide-   w.    N-(2-(3-(pyrrolidin-1-yl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   x.    N-(2-(4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)quinolin-8-yl)thiazole-4-carboxamide-   y. N-(2-p-tolylquinolin-8-yl)thiazole-4-carboxamide-   z.    N-(2-(3-fluoro-4-methylphenyl)quinolin-8-yl)thiazole-4-carboxamide-   aa. N-(2-(5-methylpyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   bb.    N-(2-(5-(methylsulfonyl)pyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide-   cc.    N-(2-(6-morpholinopyridin-3-yl)quinolin-8-yl)thiazole-4-carboxamide

Preparation ofN-(2-(2-(2-morpholinoethoxy)phenyl)quinolin-8-yl)thiazole-4-carboxamide

A solution of N-(2-(2-hydroxyphenyl)quinolin-8-yl)thiazole-4-carboxamide(0.1 g, 0.287 mmol), 4-(2-chloroethyl)morpholine (0.129 g, 0.862 mmol),and cesium carbonate (0.7 g, 2.15 mmol) in DMF (5 mL) was microwaveheated (200° C.×2 hours). The crude material was filtered and purifiedby silica gel chromatography (gradient of 0 to 90% ethyl acetate inpentane). MS (ESI) calcd for C₂₅H₂₄N₄O₃S (m/z): 460.16, found: 461[M+1].

Preparation of1-(thiazol-2-yl)-3-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)urea

Step 1) Preparation of8-isocyanato-2-(3-(trifluoromethoxy)phenyl)quinoline

2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine (252 mg, 0.830 mmol) intoluene (10 mL) was added a mixture of triphosgene (82 mg, 0.275 mmol)in toluene (5 mL). The mixture was stirred for 2 days to obtain8-isocyanato-2-(3-(trifluoromethoxy)phenyl)quinoline, which was usedwithout isolation.

Step 2) Preparation of1-(thiazol-2-yl)-3-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)urea

To a mixture of 8-isocyanato-2-(3-(trifluoromethoxy)phenyl)quinoline(0.277 mmol) in toluene (5 mL) was added 2-aminothiazole (0.553 mmol, 55mg). The mixture was concentrated to dryness, redissolved in pyridineand microwave heated (140° C.×10 min). The reaction mixture was dilutedwith CH₂Cl₂, washed with saturate aqueous NaHCO₃, water, brine. Theorganic layer was dried (Na₂SO₄) and concentrated under reducedpressure. Purification by column chromatography (0% to 100% ethylacetate in pentane) afforded the desired product. MS (ESI) calcd forC₂₀H₁₃F₃N₄O₂S (m/z): 430.07, found: 431 [M+1].

The following materials were prepared in a similar fashion:

-   a.    1-(pyridin-2-yl)-3-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)urea-   b.    1-(pyridin-3-yl)-3-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)urea

Preparation ofN-(pyridin-3-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinolin-8-amine

To a mixture of 2-(3-(trifluoromethyl)phenyl)quinolin-8-amine (200 mg,0.7 mmol), 3-pyridinecarbaldehyde (150 mg, 1.4 mmol) and acetic acid (84mg, 1.4 mmol) in methanol (10 mL) was added NaCNBH₃ (88 mg, 1.4 mmol) inportions at room temperature, then the reaction was stirred at roomtemperature for 5 h. The reaction was concentrated in vacuo, the residuewas taken up in DCM, The solution was washed with water (1×50 mL),treated with active carbon and Na₂SO₄, filtered through a silica gelpad, and the pad was washed with CH₂Cl₂. The organic solutions werecombined and concentrated in vacuo, triturated with a mixture of ethylacetate/pentane and filtered to giveN-(pyridin-3-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinolin-8-amine asa yellow solid (220 mg, yield 83%) MS (ESI) calcd for C₂₂H₁₆F₃N₃ (m/z):379.13, found: 380 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-(pyridin-2-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinolin-8-amine-   b.    N-(thiazol-2-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinolin-8-amine-   c.    N-(cyclopentylmethyl)-2-(3-(trifluoromethyl)phenyl)quinolin-8-amine-   d.    N-(pyridin-2-ylmethyl)-2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine-   e.    N-(pyridin-3-ylmethyl)-2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine-   f.    N-(thiazol-2-ylmethyl)-2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine-   g.    N-(cyclopentylmethyl)-2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine

Preparation of 2-(pyridin-3-yl)-N-(pyridin-3-ylmethyl)quinolin-8-amine

Step 1) Preparation of 2-chloro-N-(pyridin-3-ylmethyl)quinolin-8-amine

A mixture of 2-chloroquinolin-8-amine (1.75 g, 9.8 mmol),3-pyridinecarbaldehyde (2.14 g, 20 mmol) and AcOH (1.2 g, 20 mmol) inMeOH (10 mL) were added NaCNBH₃ (1.26 g, 20 mmol) in portions at roomtemperature, then the reaction mixture was stirred at room temperaturefor 5 h. The reaction was concentrated in vacuo, and the residue wastaken up in CH₂Cl₂. The solution was washed with water (1×50 mL), driedover Na₂SO₄, concentrated and purified by silica gel column (20:1pentane/ethyl acetate) to give2-chloro-N-(pyridin-3-ylmethyl)quinolin-8-amine as a yellow oil (2.1 g,yield 80%).

Step 2) Preparation of2-(pyridin-3-yl)-N-(pyridin-3-ylmethyl)quinolin-8-amine

A mixture of 2-chloro-N-(pyridin-3-ylmethyl)quinolin-8-amine (160 mg,0.59 mmol), 3-pyridylboronic acid (111 mg, 0.9 mmol), PdCl₂(dppf).CH₂Cl₂complex (53 mg, 0.06 mmol), K₂CO₃ (248 mg, 1.8 mmol) and dioxane/H₂O(5:1, 6 ml) was stirred at 80° C. for 3 h under N₂. The reaction wasfiltered, concentrated and purified by prepTLC (4:1 pentane/ethylacetate) to give 2-(pyridin-3-yl)-N-(pyridin-3-ylmethyl)quinolin-8-amineas a yellow solid (139 mg, yield 75%). MS (ESI) calcd for C₂₀H₁₆N₄(m/z): 312.14, found: 313 [M+1].

The following materials were prepared in a similar fashion:

-   a. N-(pyridin-3-ylmethyl)-2-(pyridin-4-yl)quinolin-8-amine-   b.    N-(pyridin-3-ylmethyl)-2-(2-(trifluoromethyl)phenyl)quinolin-8-amine-   c.    2-(3-(methylsulfonyl)phenyl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   d.    2-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   e.    2-(2-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   f.    2-(4-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   g.    2-(2-fluoro-5-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   h.    2-(3-fluoro-5-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   i.    N-(pyridin-3-ylmethyl)-2-(4-(trifluoromethyl)phenyl)quinolin-8-amine-   j. 2-(3-morpholinophenyl)-N-(pyridin-3-ylmethyl)quinolin-8-amine-   k.    N-(pyridin-3-ylmethyl)-2-(3-(pyrrolidin-1-yl)phenyl)quinolin-8-amine

Preparation ofN-(2-(3-(morpholinomethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide

A solution of N-(2-(3-formylphenyl)quinolin-8-yl)thiazole-4-carboxamide(54 mg, 0.150 mmol) and morpholine (37 μL, 0.450 mmol) in a mixture ofTHF (4 mL) and Ethanol (8 mL) was added Na(OAc)₃BH (95 mg, 0.450 mmol).The reaction was stirred 18 hours, and NaBH₄ (17 mg, 3 eq) and aceticacid (500 uL) and the reaction was stirred for 2 hours. The reaction wasquenched with water/methanol mixture, concentrated to dryness, anddiluted with CH₂Cl₂. The resulting solution was washed with 1N NaOH,water, brine, dried (Na₂SO₄) and concentrated. The crude material waspurified by silica gel column chromatography (0 to 10% methanol gradientin CH₂Cl₂ modified with 0.1% triethylamine). The product was lyophilizedin a mixture of acetonitrile/water to obtainN-(2-(3-(morpholinomethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide(46 mg, 71% yield). MS (ESI) calcd for C₂₄H₂₂N₄O₂S (m/z): 430.15, found:431 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-(2-(3-(pyrrolidin-1-ylmethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   b.    N-(2-(3-((dimethylamino)methyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   c. tert-butyl    4-(3-(8-(thiazole-4-carboxamido)quinolin-2-yl)benzyl)piperazine-1-carboxylate

Preparation ofN-(2-(3-(piperazin-1-ylmethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamideHydrochloride

Tert-butyl-4-(3-(8-(thiazole-4-carboxamido)quinolin-2-yl)benzyl)piperazine-1-carboxylatefrom above was treated with a mixture of 25% TFA in CH₂Cl₂ for 18 hours,concentrated to dryness. The residue was suspended in CH₂Cl₂, washedwith aqueous NaHCO₃ (sat.), dried (Na₂SO₄) and concentrated. Theresulting residue was diluted in a minimal amount of dioxane, treatedwith a slight excess of HCl in methanol, followed by diethyl ether. Theresulting HCl salt ofN-(2-(3-(piperazin-1-ylmethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamidewas collected by filtration. (24 mg 32% yield over two steps). MS (ESI)calcd for C₂₄H₂₃N₅OS (m/z): 429.16, found: 430 [M+1].

Preparation ofN-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)-N′-2-thiazolyl-sulfamide

A solution of 2-(3-(trifluoromethoxy)phenyl)quinolin-8-amine (153 mg,0.500 mmol) and triethylamine (104 μL, 1.5 eq) in anhydrous CH₂Cl₂ (10mL) was cooled to 0° C. A mixture of chlorosulfonic acid (64 mg, 1.1 eq)in CH₂Cl₂ (3 mL) was added and the reaction mixture was stirred at 0° C.for 30 minutes, warmed to room temperature and stirred for 1 hour. SolidPCl₅ (114 mg, 1.1 eq) was added, the reaction mixture was heated toreflux for 1 hour and then cooled to room temperature. The mixture wassplit into 5 equal portions by volume. To one portion was added2-aminothiazole (200 mg) and DIPEA (0.200 mL). The mixture was stirredfor 2.5 hours and water was added. The organic layer was washed withwater, brine, dried (NaSO₄) and concentrated. The residue was purifiedby Prep-HPLC, and the fractions were lyophilized to afford the productas a solid (10.9 mg, 23% yield). MS (ESI) calcd for C₁₉H₁₃F₃N₄O₃S₂(m/z): 466.04, found: 467 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)pyrrolidine-1-sulfonamide-   b.    N-[2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl]-N′-3-pyridyl-sulfamide-   c. tert-butyl    4-(N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)sulfamoyl)piperazine-1-carboxylate-   d.    N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)piperazine-1-sulfonamide:

For the preparation ofN-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)piperazine-1-sulfonamide;tert-butyl4-(N-(2-(3-(trifluoromethoxy)phenyl)quinolin-8-yl)sulfamoyl)piperazine-1-carboxylatewas deprotected using 25% TFA in CH₂Cl₂ for 3 hours, and concentratedprior to purification.

Preparation of methyl 2-amino-4-fluorobenzoate

To a solution of 2-amino-4-fluorobenzoic acid (2.0 g, 12.9 mmol) inmethanol (50 mL) was added thionyl chloride (1.8 mL, 25.8 mmol). Themixture was refluxed overnight and concentrated to dryness. The residuewas extracted with CH₂Cl₂ (30 mL), washed by aq. NaHCO₃ (20 mL), water,and brine, dried and concentrated to give methyl2-amino-4-fluorobenzoate as yellow solid (1.4 g).

Preparation of 2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylic acid

Methyl 2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylate wasprepared according to the procedure outlined by Demaude et al in Journalof Combinatorial Chemistry (2004), vol 6, p. 768-775.

Preparation of 2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylic acid

To a mixture of Methyl2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylate (1.1 g, 3.16 mmol)dissolved in THF (20 mL) was added a mixture of lithium hydroxide (227mg, 9.5 mmol) in water (15 mL). The reaction was stirred 70 hours. Thereaction mixture was concentrated to remove the THF and the aqueoussolution was adjusted to pH=1 with 4N HCl (aq). The solid was collectedby filtration, rinsed with water, and dried under vacuum to obtain2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylic acid as a tan solid(973 mg, 92% yield).

The following materials were prepared in a similar fashion:

-   a. 2-phenylquinoline-8-carboxylic acid-   b. 2-(pyridin-3-yl)quinoline-8-carboxylic acid-   c. 2-(2-chloropyridin-4-yl)quinoline-8-carboxylic acid-   d. 2-(2-(trifluoromethyl)phenyl)quinoline-8-carboxylic acid-   e. 2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxylic acid-   f. 2-(4-(trifluoromethyl)phenyl)quinoline-8-carboxylic acid-   g. 2-(5-methylisoxazol-3-yl)quinoline-8-carboxylic acid-   h. 2-(3-morpholinophenyl)quinoline-8-carboxylic acid-   i. 5-fluoro-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxylic acid-   j. 6-fluoro-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxylic acid-   k. 7-fluoro-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxylic acid

Preparation ofN-(pyridin-3-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide

To a mixture of 2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylicacid (1 gram, 3.3 mmol) and HATU (1.71 g, 4.5 mmol) in DMF (30 mL) wasadded 3-aminopyridine (423 mg, 4.5 mmol) followed by DIPEA (1.03 mL, 6mmol). The mixture was stirred 18 hours, water (150 mL) was added andthe resulting precipitate was collected by filtration. The crudematerial was purified by chromatography (silica gel, gradient 0 to 100%Ethyl acetate in Pentane), the desired fraction was concentrated and theproduct recrystallized from methanol to obtain the product as a whitesolid (550 mg, 45% yield).

MS (ESI) calcd for C₂₂H₁₄F₃N₃O₂ (m/z): 409.10, found: 410 [M+1].

The following materials were prepared in a similar fashion:

-   a. N,2-diphenylquinoline-8-carboxamide-   b. N-phenyl-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   c. N-phenyl-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   d. 2-phenyl-N-(thiazol-2-yl)quinoline-8-carboxamide-   e.    N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   f.    N-(thiazol-2-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   g. 2-phenyl-N-(pyrazin-2-yl)quinoline-8-carboxamide-   h.    N-(pyrazin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   i.    N-(pyrazin-2-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   j.    N-(pyridin-2-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   k.    N-(pyridin-4-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   l.    N-(4-methylthiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   m.    N-(1,3,4-thiadiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   n.    N-(5-methyl-1,3,4-thiadiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   o.    N-(pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   p.    N-(pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   q.    N-(pyridin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   r.    N-(5-tert-butyl-1,3,4-thiadiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   s.    N-(1H-pyrazol-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   t.    N-(pyridin-2-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   u.    N-(pyridin-3-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   v.    N-(pyridin-4-ylmethyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   w.    N-(2-oxotetrahydrofuran-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   x.    N-(tetrahydro-2H-pyran-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   y.    N-cyclopentyl-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   z.    N-(pyrimidin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   aa.    N-(5-methylthiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   bb.    N-(pyrimidin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   cc.    N-(4-methylpyrimidin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   dd.    N-(3,5-dimethylisoxazol-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ee.    N-(1,3-dimethyl-1H-pyrazol-5-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ff.    N-(4,5-dimethylthiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   gg.    N-(4,6-dimethylpyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   hh.    N-(4-phenylthiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ii.    N-(benzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   jj.    N-(5-chloropyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   kk.    N-(2-chloropyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ll.    N-(6-chloropyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   mm.    N-(3-methylisothiazol-5-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   nn.    N-(2-chloropyridin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   oo. methyl    5-(2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamido)furan-2-carboxylate-   pp.    2-(5-methylisoxazol-3-yl)-N-(pyridin-2-yl)quinoline-8-carboxamide-   qq.    2-(5-methylisoxazol-3-yl)-N-(pyridin-3-yl)quinoline-8-carboxamide-   rr.    2-(5-methylisoxazol-3-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   ss.    2-(5-methylisoxazol-3-yl)-N-(pyrimidin-4-yl)quinoline-8-carboxamide-   tt.    N-(5-methylisoxazol-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   uu.    N-(3,4-dimethylisoxazol-5-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   vv.    N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ww.    N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   xx.    N-(pyridin-3-yl)-2-(2-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   yy.    N-(pyrimidin-4-yl)-2-(2-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   zz.    N-(quinuclidin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   aaa.    N-(6-chloropyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   bbb. Ethyl    2-(2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamido)thiazole-4-carboxylate-   ccc.    N-(thiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ddd.    N-(pyridin-2-yl)-2-(4-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   eee.    N-(pyridin-3-yl)-2-(4-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   fff.    N-(pyrimidin-4-yl)-2-(4-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ggg.    N-(pyridazin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   hhh. 2-(3-morpholinophenyl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   iii. 2-(3-morpholinophenyl)-N-(pyridin-2-yl)quinoline-8-carboxamide-   jjj. 2-(3-morpholinophenyl)-N-(pyridin-3-yl)quinoline-8-carboxamide-   kkk.    2-(3-morpholinophenyl)-N-(pyrimidin-4-yl)quinoline-8-carboxamide-   lll.    N-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   mmm.    N-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   nnn.    N-(4-(morpholinomethyl)thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ooo.    N-(4-(morpholinomethyl)thiazol-2-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   ppp. 2-(pyridin-3-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   qqq. N-(pyrazin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   rrr. N-(pyridin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   sss. N,2-di(pyridin-3-yl)quinoline-8-carboxamide-   ttt.    N-(5-(morpholinomethyl)thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   uuu.    N-(5-(morpholinomethyl)thiazol-2-yl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   vvv.    N-(4-(morpholinomethyl)thiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   www.    N-(5-(morpholinomethyl)thiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   xxx. 2-(pyridin-3-yl)-N-(pyrimidin-4-yl)quinoline-8-carboxamide-   yyy.    2-(pyridin-3-yl)-N-(5-(pyrrolidin-1-ylmethyl)thiazol-2-yl)quinoline-8-carboxamide-   zzz.    N-(5-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   aaaa.    N-(4-methylthiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   bbbb.    N-(4,5-dimethylthiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   cccc.    N-(6-(pyrrolidin-1-ylmethyl)pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   dddd.    N-(6-(morpholinomethyl)pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   eeee.    2-(pyridin-3-yl)-N-(6-(pyrrolidin-1-ylmethyl)pyridin-2-yl)quinoline-8-carboxamide-   ffff.    N-(6-(morpholinomethyl)pyridin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   gggg.    N-(benzo[d]thiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   hhhh.    2-(pyridin-3-yl)-N-(1,3,4-thiadiazol-2-yl)quinoline-8-carboxamide-   iiii.    N-(5-methyl-1,3,4-thiadiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   jjjj.    N-(3-methylisothiazol-5-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   kkkk. 2-(pyridin-3-yl)-N-(pyridin-4-yl)quinoline-8-carboxamide-   llll.    N-(4-methylthiazol-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   mmmm.    N-(5-methylthiazol-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   nnnn.    N-(4,5-dimethylthiazol-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   oooo.    N-(5-methylthiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   pppp.    N-(4,6-dimethylpyridin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   qqqq.    N-(6-methylpyridin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   tttt.    N-(benzo[d]thiazol-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   ssss.    2-(3-morpholinophenyl)-N-(1,3,4-thiadiazol-2-yl)quinoline-8-carboxamide-   tttt.    N-(5-methyl-1,3,4-thiadiazol-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   uuuu.    N-(3-methylisothiazol-5-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   vvvv. 2-(3-morpholinophenyl)-N-(pyridin-4-yl)quinoline-8-carboxamide-   wwww.    N-(3-(morpholinomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   xxxx. N-(pyridazin-3-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   yyyy.    N-(5-methyl-1,3,4-oxadiazol-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   zzzz.    N-(5-fluoropyridin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   aaaaa.    N-(5-chloropyridin-2-yl)-2-(pyridin-3-yl)quinoline-8-carboxamide-   bbbbb.    N-(4,6-dimethylpyridin-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   ccccc.    N-(6-methylpyridin-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   ddddd.    2-(3-morpholinophenyl)-N-(pyridazin-3-yl)quinoline-8-carboxamide-   eeeee.    5-fluoro-N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   fffff.    5-fluoro-N-(pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ggggg.    6-fluoro-N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   hhhhh.    6-fluoro-N-(pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   iiiii.    7-fluoro-N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   jjjjj.    N-(1-methyl-1H-pyrazol-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   kkkkk.    N-(4-(morpholinomethyl)thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   lllll.    N-(5-methyl-1,3,4-oxadiazol-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   mmmmm.    N-(5-fluoropyridin-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   nnnnn.    N-(5-chloropyridin-2-yl)-2-(3-morpholinophenyl)quinoline-8-carboxamide-   ooooo.    2-(3-morpholinophenyl)-N-(pyrazin-2-yl)quinoline-8-carboxamide-   ppppp.    N-(5-(pyrrolidin-1-ylmethyl)pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   qqqqq.    N-(6-(morpholinomethyl)pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   rrrrr.    N-(6-(pyrrolidin-1-ylmethyl)pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   sssss.    N-(6-morpholinopyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   ttttt.    N-(6-(pyrrolidin-1-yl)pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   uuuuu.    N-(2-morpholinopyridin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   vvvvv.    N-(2-(pyrrolidin-1-yl)pyridin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide

Preparation of 4-chloro-2-(difluoromethyl)pyridine

To a solution of 4-chloropicolinaldehyde (1.0 grams, 7.06 mmol) inanhydrous CH₂Cl₂ (40 mL) cooled to −78° C. was added Diethylaminosulfurtrifluoride (3.7 mL, 28.2 mmol) over a 2 minute period. The solution waswarmed to room temperature and stirred for 4 hours. The reaction mixturewas cooled to 0° C., and was slowly quenched with the addition of a 1:1mixture of aqueous NaHCO₃ (sat.) and 1M NaOH. The solution was extractedwith CH₂Cl₂ (2×), and the organic layer was washed with water, brine,dried (Na₂SO₄) and concentrated to obtain a red brown oil (0.78 g, 68%yield). The product was used as is in the next step.

Preparation of2-(3-(difluoromethyl)phenyl)-N-(thiazol-2-yl)quinoline-8-carboxamide

Step 1) Preparation of2-(3-(difluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

To a solution of 1-bromo-3-(difluoromethyl)benzene (5.0 g, 24.2 mmol) inDMF (30 mL) was added bis(pinacolato)diboron (12.5 g, 50.0 mmol), KOAc(4.9 g, 50.0 mmol) and Pd(dppf)Cl₂.CH₂Cl₂ (0.5 g, 2.4 mmol). Thereaction mixture was stirred under nitrogen at 85° C. for 12 hours, thenthe reaction was cooled to room temperature and water (20 mL) was added.The mixture was extracted with ethyl acetate (3×30 mL).The combinedorganic layers were dried over Na₂SO₄ and concentrated in vacuo, theresidue was purified by silica gel column chromatography (petroleumether:ethyl acetate=150:1) to obtain2-(3-(difluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(4.5 g, 74% yield).

The following material were prepared in a similar fashion:

-   a.    2-(difluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine

Step 2) Preparation of2-(3-(difluoromethyl)phenyl)quinoline-8-carboxylic acid

To a solution of 2-chloroquinoline-8-carboxylic acid (3.1 g, 15.0 mmol)in DME (20 mL) and water (2 mL) was added2-(3-(difluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(4.5 g, 17.7 mmol), K₃PO₄ (5.2 g, 22.6 mmol) and Pd(dppf)Cl₂.CH₂Cl₂(0.50 g, 0.63 mmol). The reaction mixture was stirred under nitrogen at85° C. for 12 hours, then the reaction was cooled to room temperatureand water (20 mL) was added. The mixture was filtered and the filtercake was washed with water, and the solid was dried in vacuo to give2-(3-(difluoromethyl)phenyl)quinoline-8-carboxylic acid (3.6 g, 80%yield).

The following material were prepared in a similar fashion:

-   a. 2-(2-(difluoromethyl)pyridin-4-yl)quinoline-8-carboxylic acid

Step 3) Preparation of2-(3-(difluoromethyl)phenyl)-N-(thiazol-2-yl)quinoline-8-carboxamide

A mixture of 2-(3-(difluoromethyl)phenyl)quinoline-8-carboxylic acid(250 mg, 0.84 mmol), thiazol-2-amine (84 mg, 1.2 mmol), HATU (0.64 g,1.68 mmol) and DIPEA (0.22 g, 1.68 mmol) in DMF (25 mL) was stirred at40° C. for 12 hours. A saturated solution of NaHCO₃ (5 mL) was added andthe mixture was filtered, the residue was washed with methanol (2×5 mL)and solid was dried in vacuo to give2-(3-(difluoromethyl)phenyl)-N-(thiazol-2-yl)quinoline-8-carboxamide asa solid. (125 mg, 39% yield) MS (ESI) calcd for C₂₀H₁₃F₂N₃OS (m/z):381.07, found: 382 [M+1].

The following materials were prepared in a similar fashion:

-   a.    2-(3-(difluoromethyl)phenyl)-N-(pyridin-3-yl)quinoline-8-carboxamide-   b.    2-(3-(difluoromethyl)phenyl)-N-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)quinoline-8-carboxamide-   c.    2-(3-(difluoromethyl)phenyl)-N-(5-(pyrrolidin-1-ylmethyl)thiazol-2-yl)quinoline-8-carboxamide-   d.    2-(3-(difluoromethyl)phenyl)-N-(5-(pyrrolidin-1-ylmethyl)pyridin-2-yl)quino    line-8-carboxamide-   e.    2-(3-(difluoromethyl)phenyl)-N-(4-(morpholinomethyl)phenyl)quinoline-8-carboxamide-   f.    2-(3-(difluoromethyl)phenyl)-N-(3-(morpholinomethyl)phenyl)quinoline-8-carboxamide-   g.    2-(3-(difluoromethyl)phenyl)-N-(6-(morpholinomethyl)pyridin-2-yl)quinoline-8-carboxamide-   h.    2-(3-(difluoromethyl)phenyl)-N-(6-(pyrrolidin-1-ylmethyl)pyridin-2-yl)quinoline-8-carboxamide-   i.    2-(3-(difluoromethyl)phenyl)-N-(6-(morpholinomethyl)pyridin-3-yl)quinoline-8-carboxamide-   j.    2-(3-(difluoromethyl)phenyl)-N-(6-(pyrrolidin-1-ylmethyl)pyridin-3-yl)quinoline-8-carboxamide-   k.    2-(2-(difluoromethyl)pyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   l.    2-(2-(difluoromethyl)pyridin-4-yl)-N-(pyridin-3-yl)quinoline-8-carboxamide-   m.    2-(2-(difluoromethyl)pyridin-4-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)quinoline-8-carboxamide-   n.    2-(2-(difluoromethyl)pyridin-4-yl)-N-(5-methylthiazol-2-yl)quinoline-8-carboxamide-   o.    2-(2-(difluoromethyl)pyridin-4-yl)-N-(pyrimidin-4-yl)quinoline-8-carboxamide-   p.    2-(3-(difluoromethyl)phenyl)-N-(3-(pyrrolidin-1-ylmethyl)phenyl)quinoline-8-carboxamide

Preparation ofN-(piperidin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide

The same general amide coupling procedure detailed above was employedusing tert-butyl 4-aminopiperidine-1-carboxylate. The product wasdeprotected by treatment with 25% TFA in CH₂Cl₂ for 72 hours andconcentrated to dryness. The residue was dissolved in CH₂Cl₂, washedwith saturated aq. NaHCO₃, dried over Na₂SO₄ and concentrated. Afterchasing with pentane the product was isolated as a light brown solid (25mg, 33% yield over 2 steps). MS (ESI) calcd for C₂₂H₂₀F₃N₃O (m/z):399.16, found: 400 [M+1].

The following materials were prepared in a similar fashion:

-   a.    (S)-N-(pyrrolidin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   b.    N-(3-(piperidin-4-yloxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   c.    N-(3-(pyrrolidin-3-yloxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide

Preparation of tert-butyl 3-(3-aminophenoxy)pyrrolidine-1-carboxylate

Step 1) Preparation of tert-butyl3-(3-nitrophenoxy)pyrrolidine-1-carboxylate

To a mixture of 3-nitrophenol (4.0 g, 28.8 mmol), tert-butyl3-hydroxypyrrolidine-1-carboxylate (5.94 g, 31.7 mmol), PPh₃ (8.3 g,31.7 mmol) in THF (40 mL) at 0° C. over argon was added DEAD (5.52 g,31.7 mmol). The reaction was warmed to room temperature and stirred for18 hours. The reaction mixture was concentrated and purified by columnchromatography to obtain tert-butyl3-(3-nitrophenoxy)pyrrolidine-1-carboxylate (8.73 g, 98% yield)

Step 2) Preparation tert-butyl3-(3-aminophenoxy)pyrrolidine-1-carboxylate

To a solution of tert-butyl 3-(3-nitrophenoxy)pyrrolidine-1-carboxylate(8.73 g, 28.3 mmol) in methanol (50 mL) was added Raney Nickel (1.0 g).The solution was stirred over H₂ atmosphere (1 atm) 18 hours. Themixture was filtered, concentrated and purified by chromatography toobtain tert-butyl 3-(3-aminophenoxy)pyrrolidine-1-carboxylate as a whitesolid (5.47 g, 70% yield)

The following material was prepared in a similar fashion:

-   a. tert-butyl 4-(3-aminophenoxy)piperidine-1-carboxylate

Preparation of5-(2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamido)furan-2-carboxylicacid

A solution of methyl5-(2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamido)furan-2-carboxylate(12 mg) and NaOH (2 eq) in 50% Aqueous THF (6 mL) was stirred at roomtemperature overnight. The mixture was concentrated, adjusted to pH=1with conc. HCl and the resulting precipitate was collected byfiltration. Purification by TLC gave54243-(trifluoromethyl)phenyl)quinoline-8-carboxamido)furan-2-carboxylicacid (5 mg). MS (ESI) calcd for C₂₂H₁₃F₃N₂O₄ (m/z): 426.08, found: 427[M+1].

The following material was prepared in a similar fashion:

-   a.    2-(2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamido)thiazole-4-carboxylic    acid

Preparation of (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline

Step 1) Preparation of(R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane

3-Nitrophenol (2.0 g, 14.37 mmol) was taken up in 20 mL of anhydrous DMFalong with anhydrous potassium carbonate (4.96 g, 35.93 mmol) and(R)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane (2.55 mL, 18.68 mmol).The resulting reaction mixture was heated in the microwave reactor, withstirring, at 160° C. for 4 h. The crude reaction mixture was rinsed withwater, filtered and extracted with dichloromethane (3×15 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The resulting residue was purified by chromatographyusing ethyl acetate: pentane to obtain the desired product as anamber-colored oil (52%).

Step 2) Preparation of(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline

Under nitrogen, Fe powder (2.38 g, 42.54 mmol) and NH₄Cl (2.38 g, 42.54mmol) were combined, followed by addition of(R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane (1.8 g, 7.09mmol) and a 4:1 mixture of isopropanol:water (30 mL:10 mL). The reactionmixture was stirred under reflux for 18 h. The crude material wasfiltered through a pad of Celite and the filtrate was concentrated underreduced pressure. The resulting aqueous layer was extracted withdichloromethane (3×15 mL). The combined organic layers were dried(Na₂SO₄) and concentrated under reduced pressure to afford(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy) (1.2 g, 79% yield). Thematerial was used in the next step without any further purification.

The following materials were prepared in a similar fashion:

-   a. 3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline-   b. (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline-   c. 4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline-   d. (R)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline-   e. (S)-4((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline

Preparation ofN-(4-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide

To a mixture of 4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline (167mg, 0.750 mmol), 2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxylicacid (159 mg, 0.500 mmol), HATU (285 mg, 0.75 mmol) in NMP (5 mL) wasadded DIPEA (173 μL, 1.0 mmol). The reaction was stirred for 72 hours atroom temperature, water (5 mL) was added and the resulting precipitateswere collected by filtration and recrystallized from ethanol. The yellowsolid was treated with a mixture of 1:3 6N HCl/Dioxane (8 mL) overnight.The mixture was concentrated to dryness, triturated with water,collected by filtration, washed with water and dried to obtainN-(4-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide(191 mg, 79% yield). MS (ESI) calcd for C₂₆H₂₁F₃N₂O₄ (m/z): 482.15,found: 483 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-(4-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   b.    N-(3-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   c.    N-(3-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxamide-   d.    (S)-N-(3-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   e.    (R)-N-(3-(2,3-dihydroxypropoxy)phenyl)-2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxamide-   f.    (S)-3-(2,3-dihydroxypropoxy)-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide-   g.    (S)-6-(2,3-dihydroxypropoxy)-N-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)picolinamide

Preparation ofN-cyclopentyl-2-(3-(piperidin-4-yloxy)phenyl)quinoline-8-carboxamide

Step 1) Preparation of tert-butyl4-(3-formylphenoxy)piperidine-1-carboxylate

To a mixture of 3-hydroxybenzaldehyde (1.0 g), tert-butyl4-hydroxypiperidine-1-carboxylate (1.67 g, 1.1 eq), triphenylphosphine(2.35 g, 1.1 eq) in THF (15 mL) at 0° C. was added dropwise DEAD (6.74g, 4.75 eq). The mixture was warmed to room temperature and stirred for2 days. To the reaction mixture was added saturated NaHCO₃ (aq), and theaqueous layer was extracted with ethyl acetate (15 mL×3), the organiclayers were combined, dried (Na₂SO₄), and purified on silica gel columnchromatography (10% ethyl acetate in pentane) to obtain the desiredproduct as a light yellow oil (900 mg, 38% yield).

The following materials were prepared in a similar fashion:

-   a. (R)-3-(pyrrolidin-3-yloxy)benzaldehyde

Step 2) Preparation of2-(3-(1-(tert-butoxycarbonyl)piperidin-4-yloxy)phenyl)quinoline-8-carboxylicacid

Essentially the same procedure outlined in the preparation of2-(3-(trifluoromethoxy)phenyl)quinoline-8-carboxylic acid was used toprepare2-(3-(1-(tert-butoxycarbonyl)piperidin-4-yloxy)phenyl)quinoline-8-carboxylicacid utilizing tert-butyl 4-(3-formylphenoxy)piperidine-1-carboxylate asthe appropriate reactant.

The following material was prepared in a similar fashion:

-   a. (R)-2-(3-(pyrrolidin-3-yloxy)phenyl)quinoline-8-carboxylic acid

Step 3) Preparation ofN-cyclopentyl-2-(3-(piperidin-4-yloxy)phenyl)quinoline-8-carboxamide

The same general amide coupling procedure outlined above was employedusing2-(3-(1-(tert-butoxycarbonyl)piperidin-4-yloxy)phenyl)quinoline-8-carboxylicacid and cyclopentylamine. Purification by column chromatography (1:5ethyl acetate/pentane), followed by treatment with 4N HCl/MeOH andconcentration produced the product as a yellow solid. MS (ESI) calcd forC₂₆H₂₉N₃O₂ (m/z): 415.23, found: 416 [M+1].

The following materials were prepared in a similar fashion:

-   a.    2-(3-(piperidin-4-yloxy)phenyl)-N-(pyridin-3-ylmethyl)quinoline-8-carboxamide-   b.    (R)-N-cyclopentyl-2-(3-(pyrrolidin-3-yloxy)phenyl)quinoline-8-carboxamide-   c.    (R)-N-(pyridin-4-ylmethyl)-2-(3-(pyrrolidin-3-yloxy)phenyl)quinoline-8-carboxamide-   d.    2-(3-(piperidin-4-yloxy)phenyl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   e.    2-(3-(piperidin-4-yloxy)phenyl)-N-(pyridin-3-yl)quinoline-8-carboxamide-   f.    2-(3-(piperidin-4-yloxy)phenyl)-N-(pyrimidin-4-yl)quinoline-8-carboxamide-   g.    N-(5-methylthiazol-2-yl)-2-(3-(piperidin-4-yloxy)phenyl)quinoline-8-carboxamide

Preparation ofN-((2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methyl)cyclopentanamine

Step 1) Preparation of(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methanol

To a solution of 2-(3-(trifluoromethyl)phenyl)quinoline-8-carboxylicacid (1.5 g, 4.72 mmol) in THF (50 ml) was added LiAlH₄ (0.36 g, 9.46mmol) in portions at 0° C. The mixture was stirred at room temperatureovernight, quenched with water and concentrated to dryness. The residuewas diluted with water and extracted with ethylacetate. The combinedorganic layers were washed with brine, dried over Na₂SO₄, andconcentrated. Purification by column chromatography (1:10 ethylacetatein pentane) gave the product as a yellow oil (0.69 g, 48% yield).

Step 2) Preparation of8-(chloromethyl)-2-(3-(trifluoromethyl)phenyl)quinoline

To a solution of (2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methanol(0.67 g, 2.2 mmol) in CH₂Cl₂ (20 ml) was added SOCl₂ (0.32 ml, 4.4 mmol)dropwise at 0° C. The mixture was stirred at room temperature for 2 h,and then concentrated. The residue was purified by column chromatography(1:15 ethylacetate in pentane) to give the product as a white solid(0.668 g, 94% yield).

Step 3) Preparation ofN-((2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methyl)cyclopentanamine

A solution of 8-(chloromethyl)-2-(3-(trifluoromethyl)phenyl)quinoline(70 mg, 0.22 mmol), cyclopentylamine (0.1 ml, 1.09 mmol) and DIPEA (0.18ml, 1.09 mmol) in acetonitrile (2 ml) was stirred at 100° C. undermicrowave for 10 min. The mixture was concentrated. Purified by Prep-TLCto give a white solid (74.8 mg, 91% yield). MS (ESI) calcd forC₂₂H₂₁F₃N₂ (m/z): 370.17, found: 371 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-((2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methyl)thiazol-2-amine-   b.    N-((2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methyl)pyridin-3-amine-   c.    N-((2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)methyl)tetrahydro-2H-pyran-4-amine

Preparation ofN-(thiazol-2-yl)-3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxamide

Step 1) Preparation of 2-oxo-2-(3-(trifluoromethoxy)phenyl)acetaldehyde

To a solution of 1-(3-(trifluoromethoxy)phenyl)ethanone (5.0 g, 24.6mmol) dissolved in 1,4-dioxane (75 mL) and water (4 mL) was added SeO₂(4.38 g, 39.4 mmol) in one portion. The mixture was refluxed overnight.The mixture was filtered to remove the black precipitate. The filtratewas concentrated and purified by column chromatography (1:5 EthylAcetate/Pentane) to give2-oxo-2-(3-(trifluoromethoxy)phenyl)acetaldehyde as a yellow oil (5.3 g,98% yield).

The following material was prepared in a similar fashion:

-   a. 2-oxo-2-(3-(trifluoromethyl)phenyl)acetaldehyde

Step 2) Preparation of3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxylic acid

2-oxo-2-(3-(trifluoromethoxy)phenyl)acetaldehyde (1.0 g, 4.58 mmol) and2,3-diaminobenzoic acid (634 mg, 4.17 mmol) were dissolved in EtOH (70mL), and stirred at room temperature overnight. The volume was reducedto 30 mL, the precipitate was collected by filtration, washed withethanol, and dried under vacuum to give3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxylic acid as a graysolid (1.0 g, yield: 72% yield).

The following materials were prepared in a similar fashion:

-   a. 3-phenylquinoxaline-5-carboxylic acid-   b. 3-(3-(trifluoromethyl)phenyl)quinoxaline-5-carboxylic acid

Step 3) Preparation ofN-(thiazol-2-yl)-3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxamide

A mixture of 3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxylic acid(100 mg, 0.30 mmol), 2-aminothiazole (30 mg, 0.30 mmol), HATU (171 mg,0.45 mmol) and DIEA (116 mg, 0.9 mmol) in DMF (10 mL) was stirred atroom temperature for 2 h. Water (20 mL) was added, and the resultingprecipitate was collected by filtration and dried under vacuum to giveN-(thiazol-2-yl)-3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxamideas a solid (115.5 mg, 92% yield). MS (ESI) calcd for C₁₉H₁₁F₃N₄O₂S(m/z): 416.06, found: 417 [M+1].

The following materials were prepared in a similar fashion:

-   a. 3-Phenyl-N-(thiazol-2-yl)quinoxaline-5-carboxamide-   b. 3-Phenyl-N-(pyridin-3-yl)quinoxaline-5-carboxamide-   c. 3-Phenyl-N-(pyridin-2-yl)quinoxaline-5-carboxamide-   d.    N-(Thiazol-2-yl)-3-(3-(trifluoromethyl)phenyl)quinoxaline-5-carboxamide-   e.    N-(Pyridin-3-yl)-3-(3-(trifluoromethyl)phenyl)quinoxaline-5-carboxamide-   f.    N-(Pyridin-3-yl)-3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxamide-   g.    N-(Pyridin-2-yl)-3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxamide-   h.    N-(Pyrimidin-4-yl)-3-(3-(trifluoromethoxy)phenyl)quinoxaline-5-carboxamide-   i.    N-(Pyridin-2-yl)-3-(3-(trifluoromethyl)phenyl)quinoxaline-5-carboxamide-   j.    N-(Pyrimidin-4-yl)-3-(3-(trifluoromethyl)phenyl)quinoxaline-5-carboxamide

Preparation of methyl 2-amino-3-(aminomethyl)benzoate

Step 1) Preparation of methyl 3-(bromomethyl)-2-nitrobenzoate

To a mixture of methyl 3-methyl-2-nitrobenzoate (45.0 g, 0.23 mol) andNBS (45.0 g, 0.25 mol) in CCl₄ (1500 mL) was added AIBN (1.2 g, 7.3mmol) portion-wise under reflux. The mixture was refluxed (48 hours),and the solvent was removed in vacuo. The residue was purified by silicagel column chromatography (6% to 10% Ethyl acetate gradient in petroleumether) to obtain methyl 2-amino-3-(aminomethyl)benzoate (9.0 g, 14%yield).

Step 2) Preparation of methyl 3-(aminomethyl)-2-nitrobenzoate

To a solution of methyl 2-amino-3-(aminomethyl)benzoate (21.0 g, 76.9mmol) in CH₂Cl₂ (300 mL) was added a saturated NH₃ solution in methanol(1500 mL) at 0° C. The mixture was stirred at 5-10° C. for 12 hours. Thesolvent was removed in vacuo, and the residue was purified by silica gelcolumn chromatography (5% Methanol in CH₂Cl₂ modified with 0.5%triethylamine) to obtain methyl 3-(aminomethyl)-2-nitrobenzoate (16.0 g,87% yield).

Step 3) Preparation of methyl 2-amino-3-(aminomethyl)benzoate

A mixture of methyl 3-(aminomethyl)-2-nitrobenzoate (8.0 g, 38.3 mmol)and 5% Pd/C (0.9 g, 5%) in methanol (500 mL) was stirred at roomtemperature for 12 hours. The mixture was filtered and the filter cakewas washed with methanol. The solvent was removed in vacuo to obtainmethyl 2-amino-3-(aminomethyl)benzoate (5.0 g, 72% yield).

Preparation of 2-(pyridin-3-yl)quinazoline-8-carboxylic acid

Step 1) Preparation of methyl2-(pyridin-3-yl)-1,2,3,4-tetrahydroquinazoline-8-carboxylate

A mixture of methyl 2-amino-3-(aminomethyl)benzoate (5.0 g, 27.7 mmol),nicotinaldehyde (3.0 g, 27.7 mmol) and acetic acid (2.0 mL) in dioxane(50 mL) was stirred in the microwave for 20 minutes. The solvent wasremoved in vacuo and the residue was purified by chromatography onsilica gel (10% methanol in CH₂Cl₂) to give methyl2-(pyridin-3-yl)-1,2,3,4-tetrahydroquinazoline-8-carboxylate (4.0 g, 53%yield).

Step 2) Preparation of methyl 2-(pyridin-3-yl)quinazoline-8-carboxylate

A mixture of methyl2-(pyridin-3-yl)-1,2,3,4-tetrahydroquinazoline-8-carboxylate (4.0 g,14.8 mmol) and DDQ (5.0 g, 22.2 mmol) in CH₂Cl₂ (50 mL) was stirred atroom temperature for 24 hours. The solvent was removed in vacuo, and theresidue was purified by silica gel column chromatography (5% methanol inCH₂Cl₂) to obtain methyl 2-(pyridin-3-yl)quinazoline-8-carboxylate (3.5g, 89% yield).

Step 3) Preparation of 2-(pyridin-3-yl)quinazoline-8-carboxylic acid

A mixture of methyl 2-(pyridin-3-yl)quinazoline-8-carboxylate (3.5 g,13.2 mmol) and LiOH (0.48 g, 19.8 mmol) in 1:1 THF/H₂O (50 mL) wasstirred at 50° C. for 2 hours. The solvent was removed in vacuo andwater (20 mL) was added. Aqueous solution was adjusted to pH=3 with 1Naqueous hydrochloride solution. The mixture was extracted with ethylacetate (3×50 mL). The combined organic layers were dried (Na₂SO₄),concentrated in vacuo, and purified by silica gel column chromatography(2.5% MeOH in CH₂Cl₂) to obtain 2-(pyridin-3-yl)quinazoline-8-carboxylicacid (3.0 g, 90% yield).

The following material was prepared in a similar fashion:

-   a. 2-(3-(trifluoromethyl)phenyl)quinazoline-8-carboxylic acid-   b. 2-(3-morpholinophenyl)quinazoline-8-carboxylic acid

Preparation of2-(pyridin-3-yl)-N-(thiazol-2-yl)quinazoline-8-carboxamide

A mixture of 2-(pyridin-3-yl)quinazoline-8-carboxylic acid (250 g, 1.0mmol), thiazole-2-amine (94 mg, 1.0 mmol), HATU (760 mg, 2.0 mmol) andDIPEA (260 mg, 2.0 mmol) in DMF (15 mL) was stirred at 50° C. for 12hours. Water (20 mL) was added and the precipitate was collected byfiltration, washed with water (3×10 mL) and methanol (3×5 mL) to obtain2-(pyridin-3-yl)-N-(thiazol-2-yl)quinazoline-8-carboxamide (150 mg, 46%yield). MS (ESI) calcd for C₁₇H₁₁N₅OS (m/z): 333.07, found: 334 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinazoline-8-carboxamide-   b.    N-(pyrimidin-4-yl)-2-(3-(trifluoromethyl)phenyl)quinazoline-8-carboxamide-   c. 2-(pyridin-3-yl)-N-(thiazol-2-yl)quinazoline-8-carboxamide-   d.    N-(4-methylthiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)quinazoline-8-carboxamide-   e.    N-(pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)quinazoline-8-carboxamide-   f.    N-(pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)quinazoline-8-carboxamide-   g.    N-(4-methylthiazol-2-yl)-2-(pyridin-3-yl)quinazoline-8-carboxamide-   h. 2-(pyridin-3-yl)-N-(pyrimidin-4-yl)quinazoline-8-carboxamide-   i. N,2-di(pyridin-3-yl)quinazoline-8-carboxamide-   j. N-(pyridin-2-yl)-2-(pyridin-3-yl)quinazoline-8-carboxamide-   k. 2-(3-morpholinophenyl)-N-(thiazol-2-yl)quinazoline-8-carboxamide-   l.    N-(4-methylthiazol-2-yl)-2-(3-morpholinophenyl)quinazoline-8-carboxamide-   m. 2-(3-morpholinophenyl)-N-(pyridin-3-yl)quinazoline-8-carboxamide-   n. 2-(3-morpholinophenyl)-N-(pyridin-2-yl)quinazoline-8-carboxamide

Preparation of2-(2-(azetidin-1-yl)pyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide

Step 1) Preparation of2-(2-chloropyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide

A mixture of 2-(2-chloropyridin-4-yl)quinoline-8-carboxylic acid (285mg, 1.0 mmol), thiazol-2-amine (100 mg, 1.0 mmol), HATU (760 mg, 2.0mmol) and DIPEA (258 mg, 2.0 mmol) in DMF (10 mL) was stirred at 50° C.for 10 hours. The mixture was cooled to room temperature and water (20mL) was added. The mixture was extracted with ethyl acetate (3×25 mL),and the organic layer was dried over anhydrous Na₂SO₄, concentrated invacuo and purified by chromatography on silica gel (5% Methanol inCH₂Cl₂) to give2-(2-chloropyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide (290mg, 79% yield).

Step 2) Preparation2-(2-(azetidin-1-yl)pyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide

A mixture of2-(2-chloropyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide (200mg, 0.55 mmol), azetidine (314 mg, 5.5 mmol), CsF (84 mg, 0.55 mmol) andt-BuOK (185 mg, 1.65 mmol) in DMF (4 mL) under N₂ was microwave heated(150° C.×12 min). The reaction mixture was cooled, diluted with water,and extracted with ethyl acetate (3×10 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated in vacuo. The residuewas purified by chromatography on silica gel (10% methanol in CH₂Cl₂) toobtain of2-(2-(azetidin-1-yl)pyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamideas a solid (50 mg, 23% yield). MS (ESI) calcd for C₂₁H₁₇N₅OS (m/z):387.12, found: 388 [M+1].

The following material was prepared in a similar fashion:

-   a.    2-(2-morpholinopyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide

Preparation of 8-bromo-2-chloroquinoline

8-Bromo-2-chloroquinoline was prepared according to the procedureoutlined by Cottet et al in Eur. J. Org. Chem. (2003), vol 8, pgs.1559-1568.

Preparation of 2-chloroquinoline-8-carboxylic acid

To a solution of 8-bromo-2-chloroquinoline (14.3 g, 60 mmol) in toluene(90 mL) at −75° C. was added butyllithium in hexanes (2 mol/L, 30 mL)and the reaction mixture was kept for 20 min at −75° C. The reactionmixture was poured onto an excess of freshly crushed dry ice. Water wasadded (200 mL), and the aqueous layer was washed with ethyl acetate(3×100 mL), acidified to pH 1 with HCl (aq), and extracted with CH₂Cl₂(3×100 mL). The combined organic layers were dried, and concentrated toafford 2-chloroquinoline-8-carboxylic acid as a white solid. Yield 6.56g (53.6%).

Preparation of2-phenyl-N-(pyridin-3-yl)-3-(trifluoromethyl)quinoline-8-carboxamide

Step 1) Preparation of2-chloro-3-(trifluoromethyl)quinoline-8-carboxylic acid

2-Chloro-3-(trifluoromethyl)quinoline-8-carboxylic acid was preparedaccording to the procedure outlined by Cottet et al in Eur. J. Org.Chem. (2003), vol 8, pgs. 1559-1568.

Step 2) Preparation of2-chloro-N-(pyridin-3-yl)-3-(trifluoromethyl)quinoline-8-carboxamide

A mixture of 2-chloro-3-(trifluoromethyl)quinoline-8-carboxylic acid(165 mg, 0.60 mmol), 3-aminopyridine (73 mg, 0.78 mmol), HATU (365 mg,0.96 mmol), DIPEA (312 mg, 2.4 mmol) in CH₂Cl₂ (4 ml) was stirred atroom temperature under N₂ overnight. The reaction mixture was washedwith water (5 mL) and brine (3×5 mL). The organic solution was dried,concentrated, and purified by silica gel column chromatography (25%ethyl acetate in pentane to give2-chloro-N-(pyridin-3-yl)-3-(trifluoromethyl)quinoline-8-carboxamide asa white solid. (36 mg 65% yield.)

The following materials were prepared in a similar fashion

-   a.    2-chloro-N-(pyridin-2-yl)-3-(trifluoromethyl)quinoline-8-carboxamide-   b.    2-chloro-N-(thiazol-2-yl)-3-(trifluoromethyl)quinoline-8-carboxamide

Step 3) Preparation of2-phenyl-N-(pyridin-3-yl)-3-(trifluoromethyl)quinoline-8-carboxamide

A mixture of2-chloro-N-(pyridin-3-yl)-3-(trifluoromethyl)quinoline-8-carboxamide(136 mg, 0.39 mmol), phenylboronic acid (62 mg, 0.51 mmol),Pd(dppf)Cl₂.CH₂Cl₂ (39 mg, 0.048 mmol), K₂CO₃ (167 mg, 1.2 mmol) indioxane/H₂O (4:1, 3 ml) was heated (85° C.×2 hours) under a blanket ofNitrogen. The reaction mixture was evaporated, and the residue wastriturated with ethyl acetate. The mixture was filtered, concentratedand the residue was purified by prep. HPLC to give2-phenyl-N-(pyridin-3-yl)-3-(trifluoromethyl)quinoline-8-carboxamide asa white powder (62 mg, 41% yield). MS (ESI) calcd for C₂₂H₁₄F₃N₃O (m/z):393.11, found: 394[M+1].

The following materials were prepared in a similar fashion:

-   a.    2-phenyl-N-(pyridin-2-yl)-3-(trifluoromethyl)quinoline-8-carboxamide-   b.    2-phenyl-N-(thiazol-2-yl)-3-(trifluoromethyl)quinoline-8-carboxamide

Preparation ofN-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)pyrazine-2-carboxamide

Step 1) Preparation of2-(3-fluoro-5-(trifluoromethyl)phenyl)-8-nitroquinoline

A mixture of 2-chloro-8-nitroquinoline (0.580 g, 2.78 mmol),3-fluoro-5-(trifluoromethyl)phenylboronic acid (0.675 g, 3.22 mmol),K₃PO₄ (1.1 g, 5.2 mmol), and Pd(dppf)Cl₂.CH₂Cl₂ (0.10 g, 0.122 mmol) inDMF (3 mL) and water (1 mL) was microwave heated (125° C.×1 Hour). Themixture was filtered over celite and the celite cake was washed withethyl acetate (30 mL). The filtrate was combined with 60 mL aqueoussaturated sodium bicarbonate solution and the mixture was extracted withethyl acetate (3×20 mL). The organic layers were combined, washed withbrine, dried (MgSO₄), and concentrated to yield2-(3-fluoro-5-(trifluoromethyl)phenyl)-8-nitroquinoline. The product wasused without further purification.

The following materials were prepared in a similar fashion:

-   a. 2-(4-fluoro-3-(trifluoromethyl)phenyl)-8-nitroquinoline-   b. 8-nitro-2-(pyridin-4-yl)quinoline-   c. 8-nitro-2-(pyridin-3-yl)quinoline-   d. 2-(5-fluoropyridin-3-yl)-8-nitroquinoline

Step 2) Preparation of2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-amine

Crude 2-(3-fluoro-5-(trifluoromethyl)phenyl)-8-nitroquinoline (2.78mmol) was taken up in isopropyl alcohol (120 mL) and ammonium chloride(150 mg, 2.8 mmol) in water (20 mL) was added. The mixture was heated to90° C., iron powder (550 mg, 9.85 mmol) was added and the reaction wascontinued stirring at 90° C. 18 hours. The reaction mixture was filteredover celite and the celite cake was washed with ethyl acetate (150 mL).The filtrate was concentrated and the residue was taken up in 1N aqueousNaOH (80 mL) and extracted with ethyl acetate (3×25 mL). The organiclayers were combined, washed with brine, dried (MgSO₄), andconcentrated. The crude material was purified by flash chromatography (agradient of 0 to 100% ethyl acetate in pentane) to obtain2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-amine (0.42 g, 1.37mmol, 49% yield over two steps).

The following materials were prepared in a similar fashion:

-   a. 2-(4-fluoro-3-(trifluoromethyl)phenyl)quinolin-8-amine-   b. 2-(pyridin-4-yl)quinolin-8-amine-   c. 2-(pyridin-3-yl)quinolin-8-amine-   d. 2-(5-fluoropyridin-3-yl)quinolin-8-amine

Step 3) Preparation ofN-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)pyrazine-2-carboxamide

2-pyrazine carboxylic acid (0.065 g, 0.51 mmol) was combined with DIPEA(0.140 mL, 0.811 mmol) and HATU (0.200 g, 0.51 mmol) in 5 mL DMF. Themixture was stirred at room temperature for ten minutes, at which time2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-amine (0.12 g, 0.391mmol) was added. The mixture was stirred at room temperature for 18hours before 10 mL water was added. The resulting precipitate wascollected by filtration, and the solids were triturated in methanol toyieldN-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)pyrazine-2-carboxamideas a beige solid (0.065 g, 31% yield). MS (ESI) calcd for C₂₁H₁₂F₄N₄O(m/z): 412.09, found: 413[M+1].

The following materials were prepared in a similar fashion:

-   a. N-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide-   b.    N-(2-(4-fluoro-3-(trifluoromethyl)phenyl)quinolin-8-yl)picolinamide-   c. N-(2-(pyridin-4-yl)quinolin-8-yl)pyrazine-2-carboxamide-   d.    N-(2-(4-fluoro-3-(trifluoromethyl)phenyl)quinolin-8-yl)pyrazine-2-carboxamide-   e.    N-(2-(4-fluoro-3-(trifluoromethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   f.    N-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)picolinamide-   g.    N-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)isonicotinamide-   h.    N-(2-(4-fluoro-3-(trifluoromethyl)phenyl)quinolin-8-yl)isonicotinamide-   i.    N-(2-(3-fluoro-5-(trifluoromethyl)phenyl)quinolin-8-yl)thiazole-4-carboxamide-   j. N-(2-(4-fluoro-3-(trifluoromethyl)phenyl)quinolin-8-yl)benzamide-   k.    1-methyl-N-(2-(pyridin-3-yl)quinolin-8-yl)-1H-pyrazole-3-carboxamide-   l.    1-methyl-N-(2-(pyridin-3-yl)quinolin-8-yl)-1H-imidazole-4-carboxamide-   m.    N-(2-(5-fluoropyridin-3-yl)quinolin-8-yl)-1-methyl-1H-pyrazole-3-carboxamide-   n. 3-(2-morpholinoethoxy)-N-(2-(pyridin-3-yl)quinolin-8-yl)benzamide

Preparation of2-(5-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)quinoline-8-carboxamide

Step 1) Preparation of 2-(5-methylpyridin-3-yl)quinoline-8-carboxylicacid

A mixture of 2-chloroquinoline-8-carboxylic acid (0.600 g, 2.8 mmol),5-methylpyridin-3-ylboronic acid (0.410 g, 3.0 mmol), K₃PO₄ (1.1 g, 5.2mmol), and Pd(dppf)Cl₂.CH₂Cl₂ (0.10 g, 0.122 mmol) in DMF (3 mL) andwater (1 mL) was microwave heated (125° C.×1 Hour). The mixture wasfiltered over celite and the celite cake was washed with ethyl acetate(30 mL). The filtrate was combined with 60 mL aqueous saturated NaHCO₃solution and the mixture was extracted with ethyl acetate (3×20 mL). Theorganic layers were combined, washed with brine, dried (MgSO₄), andconcentrated. The crude material was purified by flash chromatography (agradient of 0 to 100% ethyl acetate in pentane) to obtain2-(5-methylpyridin-3-yl)quinoline-8-carboxylic acid (0.535 g, 2.02mmol).

The following material was prepared in a similar fashion:

-   a. 2-(2-methylpyridin-4-yl)quinoline-8-carboxylic acid

Step 2) Preparation of2-(5-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)quinoline-8-carboxamide

2-(5-methylpyridin-3-yl)quinoline-8-carboxylic acid (0.135 g, 0.51 mmol)was combined with DIPEA (0.140 mL, 0.811 mmol) and HATU (0.200 g, 0.51mmol) in DMF (5 mL). The mixture was stirred at room temperature for tenminutes, at which time 6-(morpholinomethyl)pyridin-2-amine (0.090 g,0.466 mmol) was added. The mixture was stirred 18 hours, and then water(10 mL) was added. The resulting precipitate was collected byfiltration, and the solids were triturated in methanol to obtain2-(5-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)quinoline-8-carboxamideas a tan solid (0.045 g, 22% yield. MS (ESI) calcd for C₂₆H₂₅N₅O₂ (m/z):439.20, found: 440 [M+1].

The following materials were prepared in a similar fashion:

-   a. 2-(2-methylpyridin-4-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   b. 2-(5-methylpyridin-3-yl)-N-(thiazol-2-yl)quinoline-8-carboxamide-   c.    2-(5-methylpyridin-3-yl)-N-(3-(morpholinomethyl)phenyl)quinoline-8-carboxamide

Preparation of 6-chloropyrido[3,2-d]pyrimidin-4-ol

Step 1) Preparation of 6-chloro-3-nitropicolinonitrile

A mixture of 2,6-dichloro-3-nitropyridine (40 g, 207 mmol) and CuCN(22.32 g, 248 mmol) in 1-methyl-2-pyrrolidinone (160 ml) was quicklyheated to 180° C. for 25 minutes. The mixture was cooled to roomtemperature and the deep brown solution was poured into ice water (1200ml) and stirred for 30 min. The aqueous solution was extracted withethyl acetate and boiling toluene, the organic layer was dried (Na₂SO₄)and concentrated under reduced pressure. The crude product was purifiedby silica gel column chromatography (5% to 25% Ethyl Acetate gradient inPentane) to give 6-chloro-3-nitropicolinonitrile as a yellow solid.(15.75 g, 41.4% yield)

Step 2) Preparation of 6-chloro-3-nitropicolinamide

A mixture of 6-chloro-3-nitropicolinonitrile (12 g, 65.4 mmol) andSnCl₂.H₂O (59 g, 262 mmol) in ethanol (144 ml) was heated to 85° C. for3 hours. The solution was concentrated under reduced pressure, water wasadded and a saturated aqueous solution of sodium bicarbonate was addeduntil pH=8. The mixture was extracted with ethyl acetate several times.The combined organic layers were dried (Na₂SO₄), and concentrated underreduce pressure to afford 6-chloro-3-nitropicolinamide in quantitativeyield.

Step 3) Preparation of 6-chloropyrido[3,2-d]pyrimidin-4-ol

A suspension of 6-chloro-3-nitropicolinamide (12 g, 70 mmol) in triethylorthoformate (490 ml) was refluxed for 3 hours. A yellow suspension wasformed which was cooled to room temperate. The precipitate was collectedby filtration, and dried under vacuum to obtain6-chloropyrido[3,2-d]pyrimidin-4-ol (10.44 g, 82% yield).

Preparation of4-chloro-6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidine

Step 1) Preparation of6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-ol

A mixture of 6-chloropyrido[3,2-d]pyrimidin-4-ol (3 g, 16.5 mmol),Cs_(s)CO₃ (16.1 g, 49.5 mmol), Pd(dppf)Cl₂.CH₂Cl₂(2.4 g) and3-fluorophenylboronic acid (4 g, 19.8 mmol) in 1,4-dioxane (180 ml) washeated to reflux for 3.5 hours. TLC showed the reaction was complete,solvent was removed and water was added. The mixture was neutralized topH=7-8, with 1N HCl, then extracted with ethyl acetate. The combinedorganic layers were dried (Na₂SO₄) and concentrated under reducepressure. The crude product was purified by silica gel columnchromatography to obtain6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-ol (2.63 g, yield52.6%).

Step 2) Preparation of4-chloro-6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidine

A solution of 6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-ol(1.2 g, 3.9 mmol) in SOCl₂ (10 mL) was refluxed for 2 hours. Thereaction was concentrated under reduce pressure, and the residue waschased with toluene (10 mL) to obtain4-chloro-6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidine as abrown solid. Used as is without further purification.

Step 3) Preparation of6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-amine

The brown solid4-chloro-6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidine obtainedfrom step 2 was added to a solution of NH₃ in propan-2-ol (50 mL, 12%),and stirred at 35° C. overnight. The reaction was concentrated underreduce pressure and the residue was purified by silica gel columnchromatography (66% Ethyl acetate in Pentane) to afford6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-amine (0.2 g, 17%yield) as a brown solid.

The following material was prepared in a similar fashion:

-   a. 6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-amine

Preparation ofN-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)picolinamide

To a solution of6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-amine (50 mg, 163mmol) in DMF (1 mL) was added HATU (124 mg, 330 mmol),pyridine-2-carboxylic acid (20 mg, 163 mmol) and DIPEA (0.07 mL, 330mmol). The mixture was stirred at room temperature for 18 hours. Thereaction mixture was treated with water and extracted with ethylacetate. The organic layer was washed with brine, dried (MgSO₄),concentrated to dryness and the crude product was purified by prep-TLC(10% Methanol in CH₂Cl₂) to affordN-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)picolinamideas a yellow solid (57 mg, 85% yield). MS (ESI) calcd for C₂₀H₁₂F₃N₅O₂(m/z): 411.09, found: 412 [M+1].

The following materials were prepared in a similar fashion:

-   a.    N-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)nicotinamide-   b.    N-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)-3-(trifluoromethyl)benzamide-   c.    3-(trifluoromethyl)-N-(6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-yl)benzamide-   d.    N-(6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-yl)thiazole-2-carboxamide-   e.    4-(pyrrolidin-1-ylmethyl)-N-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)benzamide-   f.    N-(6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-yl)thiazole-5-carboxamide-   g.    N-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)thiazole-4-carboxamide-   h.    N-(6-(3-(trifluoromethoxy)phenyl)pyrido[3,2-d]pyrimidin-4-yl)thiazole-5-carboxamide-   i.    N-(6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-yl)picolinamide-   j.    N-(6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-yl)nicotinamide-   k.    4-(pyrrolidin-1-ylmethyl)-N-(6-(3-(trifluoromethyl)phenyl)pyrido[3,2-d]pyrimidin-4-yl)benzamide

Preparation ofN-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)pyridine-2-sulfonamide

To a solution of 2-(3-(trifluoromethyl)phenyl)quinolin-8-amine (0.10 g,0.347 mmol) in CH₂Cl₂ (10 mL) was added DIPEA (0.120 mL, 0.695 mmol)followed by pyridine-2-sulfonyl chloride (0.065 g, 0.366 mmol). Themixture was stirred at room temperature for 18 hours. An aqueoussolution of saturated NaHCO₃ was added (30 mL), the resultingprecipitate was collected by filtration and washed with methanol toobtainN-(2-(3-(trifluoromethyl)phenyl)quinolin-8-yl)pyridine-2-sulfonamide(0.025 g, 17% yield).

MS (ESI) calcd for C₂₁H₁₄F₃N₃O₂S (m/z): 429.08, found: 430 [M+1].

Preparation of 3-(pyrrolidin-1-ylmethyl)aniline

1-(Bromomethyl)-3-nitrobenzene (5 g, 23.1 mmol) was taken up in 100 mLof anhydrous THF along with pyrrolidine (2.3 mL, 27.72 mmol) and K₂CO₃(4.8 g, 34.6 mmol). The reaction mixture was stirred at room temperaturefor 18 h and then filtered. The filtrate was concentrated under reducedpressure to afford 1-(3-nitrobenzyl)pyrrolidine. This material was takenup in 100 mL of absolute EtOH and 10% Pd on C (300 mg) was added. Theresulting reaction mixture was stirred at room temperature under 1 atmof hydrogen for 18 h. The mixture was then filtered through a pad ofCelite and the filtrate was concentrated under reduced pressure toafford 2.81 g of 3-(pyrrolidin-1-ylmethyl)aniline (70%).

The following materials were prepared in a similar fashion:

-   a. 3-(morpholinomethyl)aniline-   b. 4-(pyrrolidin-1-ylmethyl)aniline-   c. 4-(morpholinomethyl)aniline

Preparation of 4-(morpholinomethyl)thiazol-2-amine

Step 1) Preparation of tert-butyl 4-(hydroxymethyl)thiazol-2-ylcarbamate

Ethyl 2-aminothiazole-4-carboxylate (10.0 g, 58.1 mmol) was taken up in150 mL of anhydrous THF along with di-tert-butyl carbonate (Boc₂O, 12.67g, 58.1 mmol) along with 10 mg of 4-(dimethyl)aminopyridine (DMAP). Thereaction mixture was stirred at 50° C. for 4 h and then at roomtemperature for 18 h. It was then concentrated under reduced pressure toobtain a thick oil. Pentane was added and the resulting crystallinematerials were collected by filtration and dried to afford 10.5 g ofethyl 2-(tert-butoxycarbonylamino)thiazole-4-carboxylate. This material(10.5 g, 38.5 mmol) was dissolved in 300 mL of anhydrous THF and cooledin Dry Ice-acetonitrile bath. A solution of 1 M Super Hydride™ in THF(85 mL) was then added over a period of 10 min. The resulting reactionmixture was stirred at −45° C. for 2 h. Another portion of 1 M SuperHydride™ in THF (35 mL) was then added and the reaction mixture wasstirred for an additional 2 h at −45° C. The reaction was quenched at−45° C. by the addition of 50 mL of brine. Upon warming to roomtemperature, the reaction mixture was concentrated under reducedpressure. The resulting mixture was extracted with EtOAc. The combinedorganic layers were washed with brine, dried (Na₂SO₄) and concentratedunder reduced pressure. The resulting residue was purified bychromatography to afford 6.39 g of tert-butyl4-(hydroxymethyl)thiazol-2-ylcarbamate (72%).

The following material was prepared in a similar fashion:

-   a. tert-butyl 5-(hydroxymethyl)thiazol-2-ylcarbamate

Step 2) Preparation of 4-(morpholinomethyl)thiazol-2-amine

tert-Butyl 4-(hydroxymethyl)thiazol-2-ylcarbamate (2.0 g, 8.7 mmol) wastaken up in 25 mL of CH₂Cl₂ along with Et₃N (1.82 mL, 13.05 mmol) andcooled to 0° C. Methanesulfonyl chloride (0.85 mL, 10.88 mmol) was addedand the resulting reaction mixture was stirred at 0° C. for 60 min.Morpholine (3.0 mL, 35 mmol) was then added and the reaction mixture wasstirred at room temperature for 18 h. The reaction mixture wasconcentrated under reduced pressure. The resulting residue was taken upin EtOAc and washed with dilute aqueous NaHCO₃, brine, dried (Na₂SO₄)and concentrated under reduced pressure. This material was purified byfiltering through a short column of silica gel. The filtrate wasconcentrated to afford 1.88 g of tert-butyl4-(morpholinomethyl)thiazol-2-ylcarbamate. The Boc group was removed bytreating tert-butyl 4-(morpholinomethyl)thiazol-2-ylcarbamate with 20 mLof 25% TFA in CH₂Cl₂ for 18 h at room temperature. After all the solventhad been removed by concentrating and drying under high vacuum, theresulting residue was treated with a mixture of pentane/EtOAc to afford2.17 g 4-(morpholinomethyl)thiazol-2-amine as a white solid.

The following materials were prepared in a similar fashion:

-   a. 4-(pyrrolidin-1-ylmethyl)thiazol-2-amine-   b. 5-(morpholinomethyl)thiazol-2-amine-   c. 5-(pyrrolidin-1-ylmethyl)thiazol-2-amine

Preparation of 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine Step 1)Preparation of ethyl 6-aminopicolinate

To a solution of 2-amino-6-pyridinecarboxylic acid (6.0 g, 43.5 mmol) inethanol (150 mL) was added SOCl₂ (12.0 g, 101 mmol) at 0° C. Theresulting reaction mixture was stirred under reflux for 12 h. Uponcooling to room temperature, the reaction mixture was concentrated underreduced pressure. Enough saturated aqueous Na₂CO₃ solution was added toadjust the pH=9. The mixture was concentrated under reduced pressure anddichloromethane (150 mL) was added to the resulting residue. The mixturewas stirred vigorously at room temperature for 30 min and then filtered.The filtrate was concentrated under reduced pressure to afford ethyl6-aminopicolinate (5.5 g, 76%).

Step 2) Preparation of ethyl 6-(tert-butoxycarbonylamino)picolinate

To a solution of ethyl 6-aminopicolinate (5.5 g, 33 mmol) in t-BuOH (120mL) and acetone (40 mL) was added DMAP (0.08 g, 0.66 mmol) anddi-t-butyl dicarbonate (10.8 g, 49.5 mmol). The reaction mixture wasstirred at room temperature for 18 h. The solvent was removed byconcentration under reduced pressure and a mixture ofhexane/dichloromethane (180 mL, 3:1) was added. The resulting mixturewas cooled to −20° C. for 2 h. The resulting solids were collected byfiltration and dried to afford ethyl6-(tert-butoxycarbonylamino)picolinate (11.0 g, 91%).

Step 3) Preparation of tert-butyl 6-(hydroxymethyl)pyridin-2-ylcarbamate

To a stirred solution of ethyl 6-(tert-butoxycarbonylamino)picolinate(11.0 g, 33 mmol) in THF (120 mL) under nitrogen was added LiAlH₄ (3.80g, 100 mmol) in THF (60 mL) over a period of 30 min at 0° C. Thereaction mixture was stirred at 0° C. for 6 h and carefully quenched bythe addition of water (2.0 mL) and 10% NaOH solution (4.0 mL) at 0° C.The reaction mixture was filtered and the filtrate was dried (Na₂SO₄)and concentrated under reduced pressure. The resulting residue purifiedby chromatography (1:1 petroleum ether:ethyl acetate) to affordtert-butyl 6-(hydroxymethyl)pyridin-2-ylcarbamate (3.0 g, 41%).

Step 4) Preparation of (6-(tert-butoxycarbonylamino)pyridin-2-yl)methylmethanesulfonate

To a solution of tert-butyl 6-(hydroxymethyl)pyridin-2-ylcarbamate (3.0g, 13.4 mmol) and DIPEA (5.0 g, 40 mmol) in acetonitrile (30 mL) wasadded MsCl (2.0 g, 17.4 mmol) over a period of 30 min at 0° C. and themixture was stirred for 2 h at room temperature. The reaction wasquenched by adding saturated aqueous NaHCO₃ and extracted with ethylacetate (3×60 mL). The combined organic layers were washed with brine,dried (Na₂SO₄) and concentrated under reduced pressure to affordessentially quantitative yield of crude(6-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate.

Step 5) Preparation of tert-butyl6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate

A mixture containing (6-(tert-butoxycarbonylamino)pyridin-2-yl)methylmethanesulfonate (1.30 g, 3.2 mmol), pyrrolidine (0.46 g, 6.4 mmol) andK₂CO₃ (1.30 g, 9.6 mmol) in acetonitrile (15 mL) was stirred at roomtemperature for 12 h. Saturated aqueous NaHCO₃ was added and the mixturewas concentrated under reduced pressure. The resulting aqueous layer wasextracted with EtOAc. The combined organic layers were dried (Na₂SO₄)and concentrated under reduced pressure to afford tert-butyl6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate (0.75 g, 2.7 mmol, 62%for two steps).

Step 6) Preparation of 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine

To a solution of tert-butyl6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate (750 mg, 2.7 mmol) indichloromethane (10 mL) was added TFA (4.0 mL) at room temperature. Theresulting reaction mixture was stirred at room temperature for 6 h andthen concentrated under reduced pressure. Enough saturated aqueousNa₂CO₃ was added to the resulting residue to adjust the pH=9. Themixture was then extracted with ethyl acetate (3×25 mL). The combinedorganic layers were dried (Na₂SO₄) and concentrated under reducedpressure to afford 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine (440 mg,92%).

The following material was prepared in a similar fashion:

-   a. 6-(morpholinomethyl)pyridin-2-amine-   b. 6-(morpholinomethyl)pyridin-3-amine-   c. 5-(pyrrolidin-1-ylmethyl)pyridin-2-amine-   d. 6-(pyrrolidin-1-ylmethyl)pyridin-3-amine

Preparation of 2-(pyrrolidin-1-yl)pyridin-4-amine

A mixture of 2-chloro-4-aminopyridine (2.29 g, 17.8 mmol) andpyrrolidine (5.0 mL) was microwave heated at 200° C. for 10 min. Aftercooling to room temperature, the solid was filtered and washed withdichloromethane (10 mL×3). The filter cake was dissolved in aqueousK₂CO₃ and extracted with CH₂Cl₂ (40 mL×3). The combined organic layerswere dried over Na₂SO₄ and concentrated to obtain2-(pyrrolidin-1-yl)pyridin-4-amine (2.3 g, 79% yield).

The following materials were prepared in a similar fashion:

-   a. 2-morpholinopyridin-4-amine

Preparation of 6-(pyrrolidin-1-yl)pyridin-2-amine

A mixture of 4-chloro-2-aminopyridine (19.3 g, 0.150 mol), K₂CO₃ (41.7g, 0.30 mol) and pyrrolidine (32.0 g, 0.45 mol) in DMSO (150 mL) wasstirred at 190° C. for 10 hours. After cooling to room temperature,water (300 mL) was added and extracted with ethyl acetate (150 mL×4).The combined organic layers were washed with water (25 mL×3), dried overNa₂SO₄ and concentrated in vacuo, the residue was purified by silica gelchromatography (10:1 ethyl acetate/petroleum ether) to obtain6-(pyrrolidin-1-yl)pyridin-2-amine (9.0 g, 37% yield).

The following materials were prepared in a similar fashion:

-   a. 6-morpholinopyridin-2-amine

Example 4 Biological Activity

A mass spectrometry based assay was used to identify modulators of SIRT1activity. The mass spectrometry based assay utilizes a peptide having 20amino acid residues as follows: Ac-EE-K (biotin)-GQSTSSHSK(Ac)N1eSTEG-K(5TMR)-EE-NH2 (SEQ ID NO: 1) wherein K(Ac) is an acetylatedlysine residue and Nle is a norleucine. The peptide is labeled with thefluorophore 5TMR (excitation 540 nm/emission 580 nm) at the C-terminus.The sequence of the peptide substrate is based on p53 with severalmodifications. In addition, the methionine residue naturally present inthe sequence was replaced with the norleucine because the methionine maybe susceptible to oxidation during synthesis and purification.

The mass spectrometry assay is conducted as follows: 0.5 μM peptidesubstrate and 120 μM βNAD⁺ is incubated with 10 nM SIRT1 for 25 minutesat 25° C. in a reaction buffer (50 mM Tris-acetate pH 8, 137 mM NaCl,2.7 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.05% BSA). Test compounds may beadded to the reaction as described above. The SirT1 gene is cloned intoa T7-promoter containing vector and transformed into BL21(DE3). Afterthe 25 minute incubation with SIRT1, 10 μL of 10% formic acid is addedto stop the reaction. Reactions are sealed and frozen for later massspec analysis. Determination of the mass of the substrate peptide allowsfor precise determination of the degree of acetylation (i.e. startingmaterial) as compared to deacetylated peptide (product).

A control for inhibition of sirtuin activity is conducted by adding 1 μLof 500 mM nicotinamide as a negative control at the start of thereaction (e.g., permits determination of maximum sirtuin inhibition). Acontrol for activation of sirtuin activity is conducted using 10 nM ofsirtuin protein, with 1 μL of DMSO in place of compound, to determinethe amount of deacetylation of the substrate at a given timepoint withinthe linear range of the assay. This timepoint is the same as that usedfor test compounds and, within the linear range, the endpoint representsa change in velocity.

For the above assay, SIRT1 protein was expressed and purified asfollows. The SirT1 gene was cloned into a T7-promoter containing vectorand transformed into BL21(DE3). The protein was expressed by inductionwith 1 mM IPTG as an N-terminal His-tag fusion protein at 18° C.overnight and harvested at 30,000×g. Cells were lysed with lysozyme inlysis buffer (50 mM Tris-HCl, 2 mM Tris[2-carboxyethyl]phosphine (TCEP),10 μM ZnCl₂, 200 mM NaCl) and further treated with sonication for 10 minfor complete lysis. The protein was purified over a Ni-NTA column(Amersham) and fractions containing pure protein were pooled,concentrated and run over a sizing column (Sephadex S200 26/60 global).The peak containing soluble protein was collected and run on anIon-exchange column (MonoQ). Gradient elution (200 mM-500 mM NaCl)yielded pure protein. This protein was concentrated and dialyzed againstdialysis buffer (20 mM Tris-HCl, 2 mM TCEP) overnight. The protein wasaliquoted and frozen at −80° C. until further use.

Sirtuin modulating compounds that activated SIRT1 were identified usingthe assay described above and are shown below in Table 1. The EC_(1.5)values represent the concentration of test compounds that result in 150%activation of SIRT1. The EC_(1.5) values for the activating compoundsare represented by A (EC_(1.5)<1.0 uM), B (EC_(1.5) 1-25 uM), C(EC_(1.5)>25 uM). The percent maximum fold activation is represented byA (Fold activation >200%) or B (Fold Activation <200%). “NT” indicatesthe compound was not tested in a particular assay.

TABLE 1 Cmpd EC1.5 % Fold No. [M + H]+ Structure (μM) Act. 1 394

C NT 2 332

B A 3 327

A A 4 395

A B 5 400

A A 6 409

C NT 7 416

B A 8 411

A B 9 325

C B 10 393

B A 11 409

B B 12 332

A A 13 400

A A 14 416

A B 15 327

A B 16 395

A A 17 411

A A 19 409

C NT 20 427

C NT 21 427

C NT 22 427

C NT 23 461

C NT 24 410

A A 25 410

A A 26 410

B A 27 414

A A 28 401

A A 30 394

A A 31 394

A A 32 394

A A 34 383

B A 35 408

B B 36 408

C A 37 408

B B 38 401

B A 39 401

B B 40 385

C NT 41 395

A A 42 414

A B 43 332

C NT 44 457

A B 45 431

A B 46 425

A B 47 425

A A 48 395

A A 49 409

A A 50 412

C NT 51 411

B A 52 428

A A 53 422

A A 54 476

C NT 55 450

A A 56 428

A B 57 428

B B 58 428

A B 59 414

A A 60 428

A A 61 441

A A 62 418

C NT 63 412

C NT 64 331

C NT 65 396

C NT 66 418

C NT 67 412

A A 68 331

C NT 69 337

C NT 70 332

C NT 71 400

B A 72 386

C B 73 398

B B 74 350

B B 75 350

B A 76 333

A A 77 333

A A 78 368

C NT 79 346

A B 80 357

C NT 81 410

A A 82 412

B A 83 418

B A 84 400

B A 85 394

B B 86 394

B A 87 395

A A 88 426

C NT 89 428

C NT 90 427

B B 91 472

A A 92 416

A A 93 410

A A 94 416

B A 95 439

B A 96 400

C NT 97 394

C NT 98 394

C NT 99 395

C NT 100 395

A A 101 444

B A 102 402

B A 103 425

B A 104 386

C NT 105 380

C NT 106 410

B B 107 376

A B 108 417

A A 109 411

A A 110 411

A A 111 412

A A 112 438

C NT 113 467

C NT 114 461

C NT 115 453

B B 116 333

C NT 117 327

B B 118 327

B B 119 415

A A 120 431

A A 121 430

A A 122 389

A A 123 401

C NT 124 395

A B 125 417

C NT 126 411

B B 127 411

C NT 128 412

C NT 129 395

A B 130 396

A B 131 431

A A 132 425

A A 133 426

A A 134 445

A A 135 380

C NT 136 380

C NT 137 386

C NT 138 371

C NT 139 396

C NT 140 402

C NT 141 387

C NT 142 328

A B 143 371

C NT 144 387

C NT 145 313

C NT 146 313

C NT 147 483

A A 148 499

A A 149 483

A A 150 499

A A 151 483

A A 152 499

A A 153 411

C NT 154 412

A B 155 328

C NT 156 413

C NT 157 413

C NT 158 418

C NT 159 412

C NT 160 412

C NT 161 412

C NT 162 380

C NT 163 390

C NT 164 356

C NT 165 398

C NT 166 398

C NT 167 398

C NT 168 398

C NT 169 418

C NT 170 411

C NT 171 380

C NT 172 397

C NT 173 381

C NT 174 348

B A 175 461

B A 176 351

A A 177 499

A A 178 515

A A 179 388

A A 180 333

A B 181 328

C NT 182 327

A B 183 327

C NT 184 394

C NT 185 348

B B 186 348

B A 187 483

A A 188 499

A A 189 515

A A 190 432

A A 191 432

B A 192 400

C NT 193 394

C NT 194 412

A A 195 384

A B 196 347

A A 197 347

A A 198 347

A A 199 351

C NT 200 417

A A 201 401

C NT 202 403

A A 203 484

A A 204 399

B B 205 423

B B 206 476

C NT 207 483

A A 208 328

C NT 209 418

A B 210 412

B A 211 479

C NT 212 346

B B 213 364

A B 214 347

A A 215 436

A B 216 505

A A 217 483

A A 218 416

B A 219 483

A A 220 400

A A 221 397

A A 222 397

A A 223 347

C NT 224 383

A A 225 347

C NT 226 361

B A 227 477

A A 228 493

A A 229 411

B A 230 410

B A 231 426

A A 232 494

B A 233 383

B B 234 334

C NT 235 348

C NT 236 347

C NT 237 327

C NT 238 431

A A 239 431

A A 240 445

A A 241 347

A B 242 347

C NT 243 439

A A 244 440

A A 245 397

A A 246 418

B A 247 382

A A 248 355

A A 249 341

B A 250 467

A A 251 418

A A 252 432

A A 253 431

A A 254 411

A A 255 492

A A 256 492

A A 257 478

A A 258 463

C NT 259 402

A A 260 376

A A 261 465

A A 262 465

B A 263 328

A A 264 332

C NT 265 345

C NT 266 361

C NT 267 439

A A 268 425

A A 269 412

A A 270 401

C NT 271 396

B B 272 397

A A 273 430

C NT 274 499

A A 275 415

A B 276 416

A A 277 429

A A 278 445

A A 279 412

A A 280 334

C NT 281 415

B A 282 395

C NT 283 395

C NT 284 348

C NT 285 477

A A 286 474

A A 287 475

A A 288 459

A A 289 475

A A 290 459

A A 291 493

A A 292 477

A A 293 330

A A 294 330

B A 295 348

C NT 296 329

C NT 297 328

C NT 298 328

C NT 299 418

B B 300 402

B A 301 418

A A 302 459

A A 303 474

A A 304 383

A A 305 377

A A 306 476

A A 307 397

C NT 308 378

A A 309 418

B A 310 396

A A 311 396

B A 312 478

B A 313 432

A A 314 412

A B 315 412

A A 316 458

A A 317 455

B A 318 522

C NT 319 479

A A 320 463

A A 321 479

A A 322 463

A A

In still another embodiment of the invention, the compound is selectedfrom any one of compound numbers 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 24,25, 27, 28, 30, 31, 32, 41, 42, 44, 45, 46, 47, 48, 49, 52, 53, 55, 56,58, 59, 60, 61, 67, 76, 77, 79, 81, 87, 91, 92, 93, 100, 107, 108, 109,110, 111, 119, 120, 121, 122, 124, 129, 130, 131, 132, 133, 134, 142,147, 148, 149, 150, 151, 152, 154, 176, 177, 178, 179, 180, 182, 187,188, 189, 190, 194, 195, 196, 197, 198, 200, 202, 203, 207, 209, 213,214, 215, 216, 217, 219, 221, 222, 224, 227, 228, 231, 238, 239, 240,241, 243, 244, 245, 248, 250, 251, 252, 253, 254, 255, 256, 257, 275,278, 279, 285, 286, 287, 288, 289, 290, 291, 292, 301, 302, 303, 304,306, 308, 310, 316, 317, and 318.

EQUIVALENTS

The present invention provides among other things sirtuin-activatingcompounds and methods of use thereof. While specific embodiments of thesubject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) (www.tigr.org) and/or theNational Center for Biotechnology Information (NCBI)(www.ncbi.nlm.nih.gov).

1. A compound represented by the formula:

(III), or a salt thereof, wherein: each of Z¹¹, Z¹², Z¹³, and Z¹⁴ isindependently selected from N and CR, wherein R is selected fromhydrogen, halo, —OH, —C≡N, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂fluoro-substituted alkyl), —S—(C₁-C₂ fluoro-substituted alkyl), C₁-C₄alkyl, —(C₁-C₂ alkyl)-N(R¹⁴)(R¹⁴), —O—CH₂CH(OH)CH₂OH, —O—(C₁-C₄) alkyl,—O—(C₁-C₃) alkyl-N(R¹⁴)(R¹⁴), —N(R¹⁴)(R¹⁴), —S—(C₁-C₄) alkyl and C₃-C₇cycloalkyl; Y is selected from N and CR¹³, wherein R¹³ is selected fromhydrogen, halo, —C₁-C₄ alkyl, —O—(C₁-C₄ alkyl), and —O—(C₁-C₂fluoro-substituted alkyl); no more than two of Z¹¹, Z¹², Z¹³, Z¹⁴, and Yare N; X is selected —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†,—NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—S(O)₂—NR¹⁵-†,—NR¹⁵—S(O)₂—NH-†, —NH—C(═O)O-†, O—C(═O)—NH-†, —NH—C(═O)NH-†,—NH—C(═O)NR¹⁵-†, —NR¹⁵—C(═O)NH-†, —NH—NR¹⁵-†, —NR¹⁵—NH-†, —O—NH-†,—NH—O-†, —NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH-†, —NH—C(═NR¹⁵)-†, —C(═NR¹⁵)—NH-†,—C(═O)—NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH—C(O)-†, —NH—C(═S)—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—C(═S)—NH-†, —NH—S(O)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)—NH-†,—NH—S(O)₂—CR¹⁵R¹⁶-†, —CR—S(O)₂—NH-†, —NH—C(═O)—O—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—O—C(═O)—NH-†, —NH—C(═O)—NR¹⁴—CR¹⁵R¹⁶-†, —NH—C(═O)—CR¹⁵R¹⁶-†,and —CR¹⁵R¹⁶—NH—C(═O)—O-†,wherein † represents where X is bound to R¹¹,and: R¹⁵ and R¹⁶ are independently selected from hydrogen, C₁-C₄ alkyl,CF₃, and —(C₁-C₄ alkyl)-CF₃; R¹¹ is selected from a carbocycle and aheterocycle, wherein R¹¹ is optionally substituted with one to twosubstitutents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇cycloalkyl, C₁-C₄ fluoro-substituted alkyl, ═O, —O—R¹⁴, —S—R¹⁴, —(C₁-C₄alkyl)-N(R¹⁴)(R¹⁴), —N(R¹⁴)(R¹⁴), —O—(C₂-C₄ alkyl)-N(R¹⁴)(R¹⁴),—C(O)—N(R¹⁴)(R¹⁴), —C(O)—O—R¹⁴, and —(C₁-C₄ alkyl)-C(O)—N(R¹⁴)(R¹⁴), andwhen R¹¹ is phenyl, R¹¹ is also optionally substituted with3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy,3,4-ethylenedioxy, fluoro-substituted 3,4-ethylenedioxy, O-(saturatedheterocycle), fluoro-substituted —O-(saturated heterocycle), and C₁-C₄alkyl-substituted O-(saturated heterocycle), wherein each R¹⁴ isindependently selected from hydrogen, and —C₁-C₄ alkyl; or two R¹⁴ aretaken together with the nitrogen atom to which they are bound to form a4- to 8-membered saturated heterocycle optionally comprising oneadditional heteroatom selected from N, S, S(═O), S(═O)₂, and O, wherein:when R¹⁴ is alkyl, the alkyl is optionally substituted with one or more—OH, —O—(C₁-C₄ alkyl), fluoro, —NH₂₅—NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂,—NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂ and when two R¹⁴ are taken togetherwith the nitrogen atom to which they are bound to form a 4- to8-membered saturated heterocycle, the saturated heterocycle isoptionally substituted at a carbon atom with —OH, —C₁-C₄ alkyl, fluoro,—NH₂₅—NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or—N(CH₂CH₂OCH₃)₂; and optionally substituted at any substitutablenitrogen atom with —C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, or—(CH₂)₂—O—CH₃; and R¹² is selected from a carbocycle and a heterocyclebound to the rest of the compound through a carbon ring atom, whereinR¹² is optionally substituted with one to two substitutentsindependently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl,C₁-C₂ fluoro-substituted alkyl, —O—R¹⁴, —S—R¹⁴, —S(O)—R¹⁴, —S(O)₂—R¹⁴,—(C₁-C₄ alkyl)-N(R¹⁴)(R¹⁴), —N(R¹⁴)(R¹⁴), —O—(C₂-C₄ alkyl)-N(R¹⁴)(R¹⁴),—C(O)—N(R¹⁴)(R¹⁴), (C₁-C₄ alkyl)-C(O)—N(R¹⁴)(R¹⁴), —O—phenyl, phenyl,and a second heterocycle, and when R¹² is phenyl, R¹² is also optionallysubstituted with 3,4-methylenedioxy, fluoro-substituted3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted3,4-ethylenedioxy, or —O-(saturated heterocycle) wherein any phenyl,saturated heterocycle or second heterocycle substituent of R¹² isoptionally substituted with halo; —C≡N; C₁-C₄ alkyl, C₁-C₂fluoro-substituted alkyl, —O—(C₁-C₂ fluoro-substituted alkyl), —O—(C₁-C₄alkyl), —S—(C₁-C₄ alkyl), —S—(C₁-C₂ fluoro-substituted alkyl),—NH—(C₁-C₄ alkyl) and —N—(C₁-C₄ alkyl)₂, wherein: when Z¹¹ and Z¹³ areN, Z¹² is C—N(R¹⁴)(R¹⁴), R¹¹ is phenyl, pyridyl or thienyl and R¹² isphenyl substituted with at least one halo or —OR¹⁴, then X is not—NH—CR¹⁵R¹⁶-†; and wherein the compound is not:


2. The compound of claim 1, wherein: X is selected —NH—C(═O)-†,—C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†, —NH—S(═O)-†, —S(═O)—NH-†,—S(═O)₂—NH-†, —NH—S(O)₂—NR¹⁵-†, —NR¹⁵—S(O)₂—NH-†, —NH—C(═O)O-†,O—C(═O)—NH-†, —NH—C(═O)NH-†, —NH—C(═O)NR¹⁵-†, —NR¹⁵—C(═O)NH-†,—NH—NR¹⁵-†, —NR¹⁵—NH-†, —O—NH-†, —NH—O-†, —CR¹⁵R¹⁶—NH-†, —NH—C(═NR¹⁵)-†,—C(═NR¹⁵)—NH-†, —CR¹⁵R¹⁶—NH—C(O)-†, —NH—C(═S)—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—C(═S)—NH-†, —NH—S(O)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)—NH-†,—NH—S(O)₂—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR¹⁵R¹⁶-†,—CR¹⁵R¹⁶—O—C(═O)—NH-†, —NH—C(═O)—NR¹⁴—CR¹⁵R¹⁶-†, —NH—C(═O)—CR¹⁵R¹⁶-†,and —CR¹⁵R¹⁶—NH—C(═O)—O-†; and R¹² is selected from a carbocycle and amonocyclic heterocycle bound to the rest of the compound through acarbon ring atom, wherein R¹² is optionally substituted with one to twosubstitutents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R¹⁴, —S—R¹⁴, —S(O)—R¹⁴,—S(O)₂—R¹⁴, —(C₁-C₄ alkyl)-N(R¹⁴)(R¹⁴), —N(R¹⁴)(R¹⁴), —O—(C₂-C₄alkyl)-N(R¹⁴)(R¹⁴), —C(O)—N(R¹⁴)(R¹⁴), —(C₁-C₄ alkyl)-C(O)—N(R¹⁴)(R¹⁴),—O-phenyl, phenyl, and a second heterocycle, and when R¹² is phenyl, R¹²is also optionally substituted with 3,4-methylenedioxy,fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy,fluoro-substituted 3,4-ethylenedioxy, or —O-(saturated heterocycle),wherein any phenyl, saturated heterocycle, or second heterocyclesubstituent of R¹² is optionally substituted with halo; —C≡N; C₁-C₄alkyl, C₁-C₂ fluoro-substituted alkyl, —O—(C₁-C₂ fluoro-substitutedalkyl), —O—(C₁-C₄ alkyl), —S—(C₁-C₄ alkyl), —S—(C₁-C₂ fluoro-substitutedalkyl), —NH—(C₁-C₄ alkyl) and —N—(C₁-C₄ alkyl)₂.
 3. The compound ofclaim 1, wherein: X is —NH—CR¹⁵R¹⁶-†; and either: a. at least one ofZ¹¹, Z¹², Z¹³, Z¹⁴ or Y is N; or b. at least one of R¹¹ or R¹² is aheterocyclyl or a saturated carbocyclyl.
 4. The compound of claim 1,wherein R¹² is selected from aryl and heteroaryl.
 5. The compound ofclaim 4, wherein R¹² is selected from:

wherein R¹² is optionally further substituted.
 6. The compound of claim1, selected from any one of:


7. The compound of claim 6, wherein the compound is represented by aStructural Formulae selected from IIIa, IIIi, IIIj, IIIk, or IIIl. 8.The compound of claim 7, wherein the compound is represented byStructural Formula IIIa.
 9. The compound of claim 2, wherein X is—NH—C(═O)-† or —C(═O)—NH-†.
 10. The compound of claim 1, wherein R¹¹ isselected from:

wherein R^(H) is optionally further substituted.
 11. The compound ofclaim 10, represented by the formula:

wherein: X is selected from —NH—C(═O)-† or —C(═O)—NH-†; and R¹² isselected from phenyl and pyridyl, wherein R¹² is optionally substitutedwith one to two substitutents independently selected from halo, C₁-C₄alkyl, C₁-C₂ fluoro-substituted alkyl, —O—R¹⁴, —S(O)₂—R¹⁴, —(C₁-C₄alkyl)-N(R¹⁴)(R¹⁴), and —N(R¹⁴)(R¹⁴), and when R¹² is phenyl, R¹² isalso optionally substituted with 3,4-methylenedioxy, or O-(saturatedheterocycle).
 12. The compound of claim 11, wherein the compound isselected from any one of Compound Numbers 3, 4, 5, 8, 12, 13, 14, 15,16, 17, 24, 25, 27, 28, 30, 31, 32, 41, 42, 44, 45, 46, 47, 48, 49, 52,53, 55, 56, 58, 59, 60, 61, 67, 76, 77, 79, 81, 87, 91, 92, 93, 100,107, 108, 109, 110, 111, 119, 120, 121, 122, 124, 129, 130, 131, 132,133, 134, 142, 147, 148, 149, 150, 151, 152, 154, 176, 177, 178, 179,180, 182, 187, 188, 189, 190, 194, 195, 196, 197, 198, 200, 202, 203,207, 209, 213, 214, 215, 216, 217, 219, 221, 222, 224, 227, 228, 231,238, 239, 240, 241, 243, 244, 245, 248, 250, 251, 252, 253, 254, 255,256, 257, 275, 278, 279, 285, 286, 287, 288, 289, 290, 291, 292, 301,302, 303, 304, 306, 308, 310, 316, 317, and
 318. 13. The compound ofclaim 1 wherein the salt is a pharmaceutically acceptable salt.
 14. Apharmaceutical composition comprising: a. a compound of claim 1; or b. acompound having the formula:

wherein: i. X is —C(O)—NH—CR¹⁵R¹⁶-†; and each of Z¹¹, Z¹², Z¹³, Z¹⁴ andY is CR; or ii. X is —C(O)—NH—CR¹⁵R¹⁶-†; and R¹¹ and R¹² are eachoptionally substituted aryl; or iii. X is —NH—C(O)-†; and R¹² isbicyclic heterocycle; and a pharmaceutically acceptable carrier.
 15. Thepharmaceutical composition of claim 14, further comprising an additionalactive agent.
 16. A method for treating a subject suffering from orsusceptible to insulin resistance, a metabolic syndrome, diabetes, orcomplications thereof, or for increasing insulin sensitivity in asubject, comprising administering to the subject in need thereof acomposition of claim
 14. 17-18. (canceled)